Journal articles: 'Synthèss in vitro'

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Santosh Kumar, Badampudi, Papammagari Raveendra Reddy, Lakshmana Rao Krishna Rao Ravindranath, Aluru Raghavendra Guru Prasad, and Alvala Mallika. "Synthesis, characterization and in vitro antimicrobial evaluation of sulphonyl urea derivatives as potential inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH)." Acta Universitaria 25, no. 1 (March 2015): 12–21. http://dx.doi.org/10.15174/au.2015.658.

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Bellini, Erika, Claudio Varotto, Marco Borsò, Lorenza Rugnini, Laura Bruno, and Luigi Sanità di Toppi. "Eukaryotic and Prokaryotic Phytochelatin Synthases Differ Less in Functional Terms Than Previously Thought: A Comparative Analysis of Marchantia polymorpha and Geitlerinema sp. PCC 7407." Plants 9, no. 7 (July 20, 2020): 914. http://dx.doi.org/10.3390/plants9070914.

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This paper reports functional studies on the enzyme phytochelatin synthase in the liverwort Marchantia polymorpha and the cyanobacterium Geitlerinema sp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 µM cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise, in vivo exposure to 10–36 µM cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels in M. polymorpha were constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels in Geitlerinema sp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as in M. polymorpha and other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought.

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Slayden, R. A., R. E. Lee, J. W. Armour, A. M. Cooper, I. M. Orme, P. J. Brennan, and G. S. Besra. "Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis." Antimicrobial Agents and Chemotherapy 40, no. 12 (December 1996): 2813–19. http://dx.doi.org/10.1128/aac.40.12.2813.

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Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

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Ku, Bosung, Jae-Cheol Jeong, Benjamin N. Mijts, Claudia Schmidt-Dannert, and Jonathan S. Dordick. "Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway." Applied and Environmental Microbiology 71, no. 11 (November 2005): 6578–83. http://dx.doi.org/10.1128/aem.71.11.6578-6583.2005.

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ABSTRACT The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.

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Barekzi, Nazir, Swati Joshi, Scott Irwin, Todd Ontl, and Herbert P. Schweizer. "Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa." Microbiology 150, no. 4 (April 1, 2004): 795–803. http://dx.doi.org/10.1099/mic.0.26823-0.

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Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4′-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.

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Gohil, Dr Neha N., and Dr D. I. Brahmbhatt. "Synthesis, Characterization and In Vitro Antimicrobial Screening of Some PyrazolylPyridyl Substituted Dicoumarins." International Journal of Trend in Scientific Research and Development Volume-2, Issue-4 (June 30, 2018): 1692–705. http://dx.doi.org/10.31142/ijtsrd14420.

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Li, Sun, Zhu, Bian, Wang, and Si. "Identification of New Antifungal Agents Targeting Chitin Synthesis by a Chemical-Genetic Method." Molecules 24, no. 17 (August 29, 2019): 3155. http://dx.doi.org/10.3390/molecules24173155.

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Fungal infection is a leading cause of mortality in immunocompromised population; thus, it is urgent to develop new and safe antifungal agents. Different from human cells, fungi have a cell wall, which is composed mainly of polysaccharide glucan and chitin. The unique cell wall structure is an ideal target for antifungal drugs. In this research, a chemical-genetic method was used to isolate antifungal agents that target chitin synthesis in yeast cells. From a compound library, we isolated two benzothiazole compounds that showed greater toxicity to yeast mutants lacking glucan synthase Fks1 compared to wild-type yeast cells and mutants lacking chitin synthase Chs3. Both of them inhibited the activity of chitin synthase in vitro and reduced chitin level in yeast cells. Besides, these compounds showed clear synergistic antifungal effect with a glucan synthase inhibitors caspofungin. Furthermore, these compounds inhibited the growth of Saccharomyces cerevisiae and opportunistic pathogen Candida albicans. Surprisingly, the genome-wide mass-spectrometry analysis showed decreased protein level of chitin synthases in cells treated with one of these drugs, and this decrease was not a result of downregulation of gene transcription. Therefore, we successfully identified two new antifungal agents that inhibit chitin synthesis using a chemical-genetic method.

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Morii, Hiroyuki, Tadashi Eguchi, and Yosuke Koga. "In Vitro Biosynthesis of Ether-Type Glycolipids in the Methanoarchaeon Methanothermobacter thermautotrophicus." Journal of Bacteriology 189, no. 11 (April 6, 2007): 4053–61. http://dx.doi.org/10.1128/jb.01875-06.

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ABSTRACT The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the β-d-glucosyl-(1→6)-d-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose β-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose β-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose → MGA + UDP, and (ii) MGA + UDP-glucose → DGA + UDP. Both enzymes required the addition of K+ ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl2, in contrast to MGA synthase, which did not require Mg2+. It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg2+ requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg2+ requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg2+ increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.

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Matsuo, Shintaro, Satomi Tagawa, Yudai Matsusaki, Yuri Uchi, and Tetsuo Kondo. "Callose-synthesizing enzymes as membrane proteins of Betula protoplasts secrete bundles of β-1,3-glucan hollow fibrils under Ca2+-rich and acidic culture conditions." Holzforschung 74, no. 8 (August 27, 2020): 725–32. http://dx.doi.org/10.1515/hf-2019-0142.

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AbstractPreviously, it was reported that plant protoplasts isolated from Betula platyphylla (white birch) callus secreted bundles of hollow callose fibrils in acidic culture medium containing a high concentration of calcium ions (Ca2+). Here, the callose synthase was characterized from in situ and in vitro perspectives. Localization of callose synthases at the secreting site of callose fiber was indicated from in situ immunostaining observation of protoplasts. For in vitro analyses, membrane proteins were extracted from membrane fraction of protoplasts with a 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) treatment. The CHAPS extract aggregated in the presence of a high concentration of Ca2+, suggesting that Ca2+ may promote the arrangement of callose synthases in the plasma membrane. The callose synthase activity was dependent on pH and Ca2+, similar to the callose synthase of Arabidopsis thaliana. However, the synthesized fibril products were longer than those produced by callose synthases of herbaceous plants. This is the first insight into the specific properties of callose synthases of woody plants that secrete of callose hollow fibers.

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GURCHA, Sudagar S., Alain R. BAULARD, Laurent KREMER, Camille LOCHT, D. Branch MOODY, Walter MUHLECKER, Catherine E. COSTELLO, Dean C. CRICK, Patrick J. BRENNAN, and Gurdyal S. BESRA. "Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis." Biochemical Journal 365, no. 2 (July 15, 2002): 441–50. http://dx.doi.org/10.1042/bj20020107.

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Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.

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Fatoni, Ahmad, Ade Chika Paramita, Budi Untari, and Nurlisa Hidayati. "Chitosan-CuO Nanoparticles as Antibacterial Shigella dysenteriae: Synthesis, Characterization, and In Vitro Study." Jurnal Kimia Sains dan Aplikasi 23, no. 12 (January 9, 2021): 432–39. http://dx.doi.org/10.14710/jksa.23.12.432-439.

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The synthesis of chitosan- CuO nanoparticles was studied. This research’s aims were biosynthesis CuO nanoparticles, synthesis of chitosan-CuO nanoparticles, and used as an antibacterial agent of Shigella dysenteriae. CuO nanoparticles and chitosan-CuO nanoparticles were characterized by FTIR spectroscopy and X-ray diffraction, respectively. CuO nanoparticle was synthesized by the reaction between leaf extract of sweet star fruit (Averrhoa carambola L.) and copper sulfate pentahydrate. Chitosan-CuO nanoparticles were synthesized by a heating method. The suspension of chitosan-CuO nanoparticles was used as an antibacterial agent with a paper disk method. The result showed that the Cu-O group at CuO nanoparticles was detected at a wavenumber of 503, 619, 767, and 821 cm-1. The crystallite size of the CuO nanoparticles was 4.25 nm. Cu-O group bonded at N-H and O-H groups and detected at 3406 cm-1 from the FTIR spectra of chitosan-CuO nanoparticles. The average inhibition zone of chitosan-CuO nanoparticles at concentration 2.500, 5.000, 7.500, and 10.000 ppm to Shigella dysenteriae were 13.57 ± 1.55; 14.90 ± 1.20; 15.97 ± 0.76 and 17.03 ± 1.80 mm, respectively.

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Eichel, Johannes, Ying-Ying Chang, Dieter Riesenberg, and John E. Cronan. "Effect of ppGpp on Escherichia coliCyclopropane Fatty Acid Synthesis Is Mediated through the RpoS Sigma Factor (ςS)." Journal of Bacteriology 181, no. 2 (January 15, 1999): 572–76. http://dx.doi.org/10.1128/jb.181.2.572-576.1999.

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ABSTRACT Strains of Escherichia coli carrying mutations at therelA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3′,5′-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification. We report that the effect of RelA on CFA synthesis is indirect. In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity. TherelA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase.

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Zhao, Conghui, Zhenming Yu, Jaime A. Teixeira da Silva, Chunmei He, Haobin Wang, Can Si, Mingze Zhang, Danqi Zeng, and Jun Duan. "Functional Characterization of a Dendrobium officinale Geraniol Synthase DoGES1 Involved in Floral Scent Formation." International Journal of Molecular Sciences 21, no. 19 (September 23, 2020): 7005. http://dx.doi.org/10.3390/ijms21197005.

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Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.

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Kelly, Amélie A., Barbara Kalisch, Georg Hölzl, Sandra Schulze, Juliane Thiele, Michael Melzer, Rebecca L. Roston, Christoph Benning, and Peter Dörmann. "Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana." Proceedings of the National Academy of Sciences 113, no. 38 (September 6, 2016): 10714–19. http://dx.doi.org/10.1073/pnas.1609184113.

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Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

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Crick, Dean C., Mark C. Schulbach, Erin E. Zink, Marco Macchia, Silvia Barontini, Gurdyal S. Besra, and Patrick J. Brennan. "Polyprenyl Phosphate Biosynthesis inMycobacterium tuberculosis and Mycobacterium smegmatis." Journal of Bacteriology 182, no. 20 (October 15, 2000): 5771–78. http://dx.doi.org/10.1128/jb.182.20.5771-5778.2000.

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ABSTRACT Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes ω,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes ω,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the ω,E,Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the ω,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, ω,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P ofM. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.

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Holtzapple, Erik, and Claudia Schmidt-Dannert. "Biosynthesis of Isoprenoid Wax Ester in Marinobacter hydrocarbonoclasticus DSM 8798: Identification and Characterization of Isoprenoid Coenzyme A Synthetase and Wax Ester Synthases." Journal of Bacteriology 189, no. 10 (March 9, 2007): 3804–12. http://dx.doi.org/10.1128/jb.01932-06.

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ABSTRACT Marinobacter hydrocarbonoclasticus DSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described from Acinetobacter sp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain, M. aquaeolei VT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizing Marinobacter strain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of the Marinobacter wax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.

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Lu, Qiuhe, Jing Han, Ligang Zhou, Jian Zhou, and Hua Xiang. "Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei." Journal of Bacteriology 190, no. 12 (April 11, 2008): 4173–80. http://dx.doi.org/10.1128/jb.00134-08.

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ABSTRACT The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE Hme and phaC Hme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.

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Sampath, K., and C. Jayabalakrishnan. "Ruthenium(III) Thiosemicarbazone Complexes: Synthesis, Characterization, DNA Binding, Antibacterial, In vitro Anticancer and Antioxidant Studies." DJ Journal of Engineering Chemistry and Fuel 1, no. 1 (January 2, 2016): 40–53. http://dx.doi.org/10.18831/djchem.org/2016011004.

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Ramos, Itzel, E. I. Vivas, and D. M. Downs. "Mutations in the Tryptophan Operon Allow PurF-Independent Thiamine Synthesis by Altering Flux In Vivo." Journal of Bacteriology 190, no. 3 (June 8, 2007): 815–22. http://dx.doi.org/10.1128/jb.00582-07.

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ABSTRACT Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

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Dolan, Jennifer M., Jason B. Weinberg, Edmund O'Brien, Anya Abashian, Megan C. Procario, David M. Aronoff, Leslie J. Crofford, Marc Peters-Golden, Lindsay Ward, and Peter Mancuso. "Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 11 (June 1, 2016): L1111—L1120. http://dx.doi.org/10.1152/ajplung.00220.2015.

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The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections.

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Voiniciuc, Cătălin, Murali Dama, Niklas Gawenda, Fabian Stritt, and Markus Pauly. "Mechanistic insights from plant heteromannan synthesis in yeast." Proceedings of the National Academy of Sciences 116, no. 2 (December 24, 2018): 522–27. http://dx.doi.org/10.1073/pnas.1814003116.

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Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of β-(1–4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.

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Wiesmann, Kirsten E. H., Jesus Cortés, Murray J. B. Brown, Annabel L. Cutter, James Staunton, and Peter F. Leadlay. "Polyketide synthesis in vitro on a modular polyketide synthase." Chemistry & Biology 2, no. 9 (September 1995): 583–89. http://dx.doi.org/10.1016/1074-5521(95)90122-1.

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Kudlicka, Krystyna, and R. M. Brown, Jr. "Cellulose biosynthesis in higher plants." Acta Societatis Botanicorum Poloniae 65, no. 1-2 (2014): 17–24. http://dx.doi.org/10.5586/asbp.1996.003.

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Knowledge of the control and regulation of cellulose synthesis is fundamental to an understanding of plant development since cellulose is the primary structural component of plant cell walls. In vivo, the polymerization step requires a coordinated transport of substrates across membranes and relies on delicate orientations of the membrane-associated synthase complexes. Little is known about the properties of the enzyme complexes, and many questions about the biosynthesis of cell wall components at the cell surface still remain unanswered. Attempts to purify cellulose synthase from higher plants have not been successful because of the liability of enzymes upon isolation and lack of reliable in vitro assays. Membrane preparations from higher plant cells incorporate UDP-glucose into a glucan polymer, but this invariably turns out to be predominantly β -1,3-linked rather than β -1,4-linked glucans. Various hypotheses have been advanced to explain this phenomenon. One idea is that callose and cellulose-synthase systems are the same, but cell disruption activates callose synthesis preferentially. A second concept suggests that a regulatory protein as a part of the cellulose-synthase complex is rapidly degraded upon cell disruption. With new methods of enzyme isolation and analysis of the in vitro product, recent advances have been made in the isolation of an active synthase from the plasma membrane whereby cellulose synthase was separated from callose synthase.

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Abalenikhina, Yu V., and M. A. Fomina. "Protein oxidative modification and cathepsin H activity in rats’ thymocytes at nitrogen oxide (II) synthesis modulation in vitro." Kazan medical journal 95, no. 4 (August 15, 2014): 553–57. http://dx.doi.org/10.17816/kmj1842.

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Aim. To study the influence of substrate for nitrogen oxide (II) synthesis - L-arginine - and non-selective NO-synthase inhibitor - N-nitro-L-arginine-methyl ester - on protein oxidative modification in combination with rats’ thymocytes cathepsin H activity estimation in vitro. Methods. The study was performed on male Wistar rats with body weight of 280-320 g. Freshly-separated thymocytes were incubated in vitro in the full nutrient medium containing 5 мМ of N-nitro-L-arginine-methyl ester (n=8) or 5 мМ of L-arginine for 24 hours at the temperature of 37 °C (n=8). Control group consisted of thymocytes incubated in the same conditions in the full nutrient medium (n=8) for 24 hours. Nitric oxide metabolites levels were measured by spectrophotometry in the visible spectrum using the reaction with Griess reagent. Cathepsin H activity was estimated by BarrettKirschke spectrofluorimetry. Protein oxidative modification was measured by R.L. Levine method in E.E. Dubinina modification followed by carbonyl derivatives absorption spectrum quantitative analysis. Results. In nitrogen oxide (II) synthesis deficiency model, protein oxidative modification degree increased, mainly due to basic and neutral aldehyde- and ketone-dinitrophenylhydrazones level increase. Those changes were accompanied by increased activity of cathepsin H. In nitrogen oxide (II) synthesis stimulation model, level of oxidative-modified proteins decreased, mainly due to lower levels of neutral amino acid derivatives, cathepsin H activity didn’t change. Conclusion. In vitro nonselective inhibitor of inducible NO-synthase - N-nitro-L-arginine-methyl ester - stimulates protein oxidative modification and increases activity of cathepsin Н; substrate of NO synthesis - L-arginine - showes antioxidant effect.

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Mohite, Bhavna V., and Satish V. Patil. "Investigation of Bacterial Cellulose Biosynthesis Mechanism in Gluconoacetobacter hansenii." ISRN Microbiology 2014 (March 16, 2014): 1–7. http://dx.doi.org/10.1155/2014/836083.

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The present study explores the mechanism of cellulose biosynthesis in Gluconoacetobacter hansenii. The cellulose synthase enzyme was purified as membrane fraction and solubilized by treatment with 0.1% digitonin. The enzyme was separated by native-gel electrophoresis and β-D-glucan analysis was carried out using in vitro gel assay. The cellulose synthase has glycoprotein nature and composed two polypeptide subunits of 93 KDa and 85 KDa. The confirmation of β-1,4-glucan (cellulose) was performed in whole and hydrolyzed monomeric sugar form. Tinopal and Congo red were used for cellulose detection on the gel. Thus the in vitro cellulose synthesis assay with cell free enzyme fraction was attempted to improve the understanding of cellulose biosynthesis.

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Akila, Ekamparam, Markandan Usharani, and Rangappan Rajavel. "Novel Metal-based Pharmacologically Dynamic Agents of Transition Metal(II) Complexes: Designing, Synthesis, Structural Elucidation, Efficient Nuclease Activity, In Vitro Antibacterial and In Vitro Antioxidant Assay." Indian Journal of Applied Research 3, no. 5 (October 1, 2011): 60–61. http://dx.doi.org/10.15373/2249555x/may2013/16.

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Bloch, G., J. R. Chase, D. B. Meyer, M. J. Avison, G. I. Shulman, and R. G. Shulman. "In vivo regulation of rat muscle glycogen resynthesis after intense exercise." American Journal of Physiology-Endocrinology and Metabolism 266, no. 1 (January 1, 1994): E85—E91. http://dx.doi.org/10.1152/ajpendo.1994.266.1.e85.

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Time courses of the glycogen synthesis rate and of the glucose 6-phosphate (G-6-P) concentration after an electrically induced exercise were followed in the anesthetized rat gastrocnemius by in vivo 13C and 31P nuclear magnetic resonance (NMR) spectroscopy, respectively. The ratio of glycogen synthase I to glycogen synthase I and D (I/I+D) and allosteric activation by G-6-P were also studied in vitro on muscles sampled at rest and 10 min (early recovery) and 100 min (late recovery) after exercise. From early recovery to late recovery, the in vivo glycogen synthesis rate dropped from 0.46 +/- 0.06 to 0.11 +/- 0.04 mmol.kg wet tissue-1.min-1, the G-6-P concentration from 0.83 +/- 0.08 to 0.32 +/- 0.05 mmol/kg wet tissue, and I/I+D from 83 +/- 4 to 47 +/- 1%. The combination of the changes in G-6-P concentration and in I/I+D quantitatively describes the fourfold decrease in glycogen synthesis rate from early to late recovery. These results demonstrate that phosphorylation, determining glycogen synthase I/I+D, and allosteric control of glycogen synthase by G-6-P contribute approximately equally to the regulation of the postexercise in vivo glycogen synthesis rate.

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Lahiri, Sujoy, Hyejung Park, Elad L. Laviad, Xuequan Lu, Robert Bittman, and Anthony H. Futerman. "Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner." Journal of Biological Chemistry 284, no. 24 (April 8, 2009): 16090–98. http://dx.doi.org/10.1074/jbc.m807438200.

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FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (<200 nm) sphinganine concentrations, consistent with FTY720 acting as an uncompetitive inhibitor toward sphinganine. Finally, electrospray ionization-tandem mass spectrometry demonstrated, unexpectedly, elevated levels of ceramide, sphingomyelin, and hexosylceramides after incubation with FTY720. Our data suggest a novel mechanism by which FTY720 might mediate some of its biological effects, which may be of mechanistic significance for understanding its mode of action.

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McNulty, P. H., C. Ng, W. X. Liu, D. Jagasia, G. V. Letsou, J. C. Baldwin, and R. Soufer. "Autoregulation of myocardial glycogen concentration during intermittent hypoxia." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 271, no. 2 (August 1, 1996): R311—R319. http://dx.doi.org/10.1152/ajpregu.1996.271.2.r311.

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During hypoxia, the heart consumes glycogen to generate ATP. Tolerance of repetitive hypoxia logically requires prompt replenishment of glycogen, a process whose regulation is not fully understood. To examine this, we imposed a defined hypoxic stimulus on the rat heart while varying its workload. In intact rats, hypoxia reduced myocardial glycogen approximately 30% and increased both the fraction of glycogen synthase in its physiologically active (GS I) form (from 0.24 +/- 0.06 to 0.82 +/- 0.07; P < 0.005) and glycogen synthesis (from 0.087 +/- 0.011 to 0.375 +/- 0.046 mumol.g-1.min-1; P < 0.005). Reducing cardiac work (with propranolol or heterotopic transplantation) reduced glycogen breakdown, glycogen synthase activation, and glycogen synthesis in parallel, stepwise fashion in intact rats. Correspondingly, hypoxia increased GS I activity in the perfused heart in vitro, but only under conditions where glycogen was consumed. This suggests myocardial glycogen synthase is activated by systemic hypoxia and catalyzes rapid posthypoxic glycogen synthesis. Hypoxic glycogen synthase activation appears to be a proportionate, wholly intrinsic response to local glycogenolysis, operating to preserve myocardial glycogen stores independent of any extracardiac mediator of carbohydrate metabolism.

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Sivakumaran, K., M. Hema, and C. Cheng Kao. "Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts." Journal of Virology 77, no. 10 (May 15, 2003): 5703–11. http://dx.doi.org/10.1128/jvi.77.10.5703-5711.2003.

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ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

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WINTER, Elke, Monika BRUMMEL, Ricardo SCHUCH, and Friedrich SPENER. "Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis." Biochemical Journal 321, no. 2 (January 15, 1997): 313–18. http://dx.doi.org/10.1042/bj3210313.

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In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalents NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.

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Selvaggini, Serena, Carol A. Munro, Serge Paschoud, Dominique Sanglard, and Neil A. R. Gow. "Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae." Microbiology 150, no. 4 (April 1, 2004): 921–28. http://dx.doi.org/10.1099/mic.0.26661-0.

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Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

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FRANCH, Jesper, Rune ASLESEN, and Jørgen JENSEN. "Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase." Biochemical Journal 344, no. 1 (November 8, 1999): 231–35. http://dx.doi.org/10.1042/bj3440231.

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We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen synthesis. Surprisingly, adrenaline did not inhibit glycogen synthesis stimulated by glycogen-depleting contractile activity. In agreement with this, the fractional activity of glycogen synthase was high when adrenaline was present after exercise, whereas adrenaline decreased the fractional activity of glycogen synthase to a low level during stimulation with insulin. Furthermore, adrenaline activated glycogen phosphorylase almost completely during stimulation with insulin, whereas a much lower activation of glycogen phosphorylase was observed after contractile activity. Thus adrenaline does not inhibit contraction-stimulated glycogen synthesis.

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34

Palasopoulou, M., C. Haitoglou, G. Papageorgiou, and A. Dimitriadou. "PLP Induced Inhibition of Thymidylate Synthase Activity in T Lymphocytes." European Journal of Inflammation 1, no. 2 (May 2003): 79–83. http://dx.doi.org/10.1177/1721727x0300100205.

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The present study was undertaken in order to examine whether pyridoxine may be used as an inhibitor of thymidylate synthase, and thus, as an antiproliferative agent. It has already been shown by others that pyridoxal phosphate binds thymidylate synthase in vitro. Phytohemagglutinin-stimulated T lymphocytes were cultured in the presence of pyridoxine in various concentrations. Enzyme activity was measured in supernatants of sonicated cell extracts. Our results show that pyridoxine effectively inhibits thymidylate synthase (up to 60–70%) and therefore DNA synthesis (up to 42%) and cell division. We also present evidence that pyridoxine must be phosphorylated to PLP in order to exert its inhibitory effect. Our results suggest that pyridoxal phosphate, in addition to its function as a coenzyme, may have a role in regulating DNA synthesis through modulation of thymidylate synthase activity. Our findings also indicate that pyridoxal phosphate might be tested for its potential immunosuppressive and/or chemotherapeutic action, alone or in combination with other antiproliferative agents.

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Biczkowski-Gudarzi, Martina, and Frank Dittmann. "In vitro Protein Synthesis in Microinjected or Isolated Previtellogenic Oocytes of the Bug Dysdercus intermedius (Heteroptera: Pyrrhocoridae)." Entomologia Generalis 13, no. 1-2 (May 1, 1988): 9–14. http://dx.doi.org/10.1127/entom.gen/13/1988/9.

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McCool, Gabriel J., and Maura C. Cannon. "PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium." Journal of Bacteriology 183, no. 14 (July 15, 2001): 4235–43. http://dx.doi.org/10.1128/jb.183.14.4235-4243.2001.

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ABSTRACT Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to thephaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaCBm) and PhaR (PhaRBm) for activity in vivo and in vitro. PhaCBm showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaRBm had no obvious homology to PhaE. Previously we showed that PhaCBm, and here we show that PhaRBm, is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaCBm to produce an active PHA synthase.

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Geisel, J., J. A. Cook, S. H. Ashton, W. C. Wise, and P. V. Halushka. "Endotoxin tolerance is associated with decreased prostaglandin H synthases-1 and -2." American Journal of Physiology-Cell Physiology 267, no. 4 (October 1, 1994): C1067—C1072. http://dx.doi.org/10.1152/ajpcell.1994.267.4.c1067.

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Lipopolysaccharide (LPS) induces macrophage protein and eicosanoid synthesis. Previous studies have shown that LPS induction of eicosanoid metabolism is transcription dependent and that prostaglandin (PG) H synthase is the committed step in the conversion of arachidonic acid (AA) to thromboxane (Tx) B2. LPS tolerance induced by sublethal in vivo injections of LPS renders rats resistant to LPS in vivo and macrophages refractory to LPS-stimulated eicosanoid synthesis in vitro. This study examined potential activity and content changes in constitutive and mitogen inducible PGH synthase in LPS-stimulated control and tolerant macrophages. TxB2 levels were measured to evaluate basal PGH synthase activity and stimulation by Salmonella enteritidis LPS (50 micrograms/ml), calcium ionophore A-23187, and AA. All tolerant macrophage groups demonstrated decreased TxB2 synthesis. TxB2 synthesis stimulated by AA in tolerant cells was decreased by 70% (P < 0.05) compared with control macrophages. In subsequent studies changes in PGH synthase content were examined in rat peritoneal macrophages from tolerant and control rats incubated with and without LPS. Immunoblot analysis of PGH synthase-1 (constitutive) demonstrated no increase in cells stimulated with LPS compared with basal but was diminished by 62 +/- 9% (n = 4, P < 0.05) in tolerant macrophages compared with control cells. Immunoblot analysis of PGH synthase-2 (mitogen inducible) demonstrated induction in response to LPS that was maximal between 12 and 24 h. PGH synthase-2 was also induced in tolerant macrophages in response to LPS but was less than in control cells. The results demonstrate that endotoxin tolerance is associated with reduced activity and content of PGH synthase-1 and a decreased LPS induction of PGH synthase-2.(ABSTRACT TRUNCATED AT 250 WORDS)

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Scala, V., E. Camera, M. Ludovici, C. Dall'Asta, M. Cirlini, P. Giorni, P. Battilani, et al. "Fusarium verticillioides and maize interaction in vitro: relationship between oxylipin cross-talk and fumonisin synthesis." World Mycotoxin Journal 6, no. 3 (August 1, 2013): 343–51. http://dx.doi.org/10.3920/wmj2012.1527.

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Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the pathogen oxylipin profile with the fumonisin synthesis. Notably, a modification of the oxylipin profile of the pathogen during its growth on cracked maize can be demonstrated. The switch in oxylipin synthesis could indicate that the ‘presence’ of maize determinants (e.g. plant cell wall fragments and/or lipids) was able to promote the modification of the pathogen lifestyle, also by adapting the secondary metabolism, notably fumonisin synthesis.

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Huang, Dongqing, Jason Moffat, Wayne A. Wilson, Lynda Moore, Christine Cheng, Peter J. Roach, and Brenda Andrews. "Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10." Molecular and Cellular Biology 18, no. 6 (June 1, 1998): 3289–99. http://dx.doi.org/10.1128/mcb.18.6.3289.

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ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

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Shibata, Y., DR Bjorkman, M. Schmidt, Y. Oghiso, and A. Volkman. "Macrophage colony-stimulating factor-induced bone marrow macrophages do not synthesize or release prostaglandin E2." Blood 83, no. 11 (June 1, 1994): 3316–23. http://dx.doi.org/10.1182/blood.v83.11.3316.3316.

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Abstract Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

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Shibata, Y., DR Bjorkman, M. Schmidt, Y. Oghiso, and A. Volkman. "Macrophage colony-stimulating factor-induced bone marrow macrophages do not synthesize or release prostaglandin E2." Blood 83, no. 11 (June 1, 1994): 3316–23. http://dx.doi.org/10.1182/blood.v83.11.3316.bloodjournal83113316.

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Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

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Pargney, Jean-Claude. "Effets d'une plasmolyse prolongée sur les tubes polliniques angiospermiens en culture in vitro : étude ultrastructurale." Canadian Journal of Botany 66, no. 1 (January 1, 1988): 108–15. http://dx.doi.org/10.1139/b88-016.

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In angiosperm plants subjected to plasmolysis, pollen tubes may undergo substantial ultrastructural changes accompanied by a gradual deterioration of those processes involved in cell syntheses. However, some tubes quickly regenerate a polysaccharide wall and thus ensure their extension. Others undergo fragmentation of their cytoplasm and a serious breakdown in processes involved in cell wall synthesis. In these extreme cases, the endoplasmic reticulum is the only compartment that is readily discernible.

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Alfonso Pecchio, Adolfo R., Andrés M. Cardozo Gizzi, Marianne L. Renner, María Molina-Calavita, and Beatriz L. Caputto. "c-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides." Molecular Biology of the Cell 22, no. 24 (December 15, 2011): 4716–25. http://dx.doi.org/10.1091/mbc.e11-03-0259.

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The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

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STONE, Scot J., and Jean E. VANCE. "Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2." Biochemical Journal 342, no. 1 (August 10, 1999): 57–64. http://dx.doi.org/10.1042/bj3420057.

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Phosphatidylserine (PtdSer) is synthesized in mammalian cells by two base-exchange enzymes: PtdSer synthase (PSS)-1 primarily uses phosphatidylcholine as a substrate for exchange with serine, whereas PSS2 uses phosphatidylethanolamine (PtdEtn). We previously expressed murine PSS1 in McArdle hepatoma cells. The activity of PSS1 in vitro and the synthesis of PtdSer and PtdSer-derived PtdEtn were increased, whereas PtdEtn synthesis from the CDP-ethanolamine pathway was inhibited [Stone, Cui and Vance (1998) J. Biol. Chem. 273, 7293-7302]. We have now cloned and stably expressed a murine PSS2 cDNA in McArdle cells and M.9.1.1 cells [which are ethanolamine-requiring mutant Chinese hamster ovary (CHO) cells defective in PSS1]. Expression of the PSS2 in M.9.1.1 cells reversed the ethanolamine auxotrophy. However, the PtdEtn content was not normalized unless the culture medium was supplemented with ethanolamine. In both M.9.1.1 and hepatoma cells transfected with PSS2 cDNA the rate of synthesis of PtdSer and PtdSer-derived PtdEtn did not exceed that in parental CHO cells or control McArdle cells respectively, in contrast to cells expressing similar levels of murine PSS1. These observations suggest that PtdSer synthesis via murine PSS2, but not PSS1, is regulated by end-product inhibition. Moreover, expression of murine PSS2 in McArdle cells did not inhibit PtdEtn synthesis via the CDP-ethanolamine pathway, whereas expression of similar levels of PSS1 activity inhibited this pathway by approx. 50%. We conclude that murine PSS1 and PSS2, which are apparently derived from different genes, independently modulate phospholipid metabolism. In addition, mRNAs encoding the two synthases are differentially expressed in several murine tissues, supporting the idea that PSS1 and PSS2 might perform unique functions.

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Azpiazu, Iñaki, Jill Manchester, Alexander V. Skurat, Peter J. Roach, and John C. Lawrence. "Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers." American Journal of Physiology-Endocrinology and Metabolism 278, no. 2 (February 1, 2000): E234—E243. http://dx.doi.org/10.1152/ajpendo.2000.278.2.e234.

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The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [14C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.

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Solomon, Peter S., Cordula I. Jörgens, and Richard P. Oliver. "δ-Aminolaevulinic acid synthesis is required for virulence of the wheat pathogen Stagonospora nodorum." Microbiology 152, no. 5 (May 1, 2006): 1533–38. http://dx.doi.org/10.1099/mic.0.28556-0.

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δ-Aminolaevulinic acid (ALA) is synthesized in fungi by ALA synthase, a key enzyme in the synthesis of haem. The requirement for ALA synthase in Stagonospora nodorum to cause disease in wheat was investigated. The single gene encoding ALA synthase (Als1) was cloned and characterized. Expression analysis determined that Als1 transcription was up-regulated during germination and also towards the latter stages of the infection. The Als1 gene was further characterized by homologous gene replacement. The inactivation of Als1 resulted in strains producing severely stunted germ tubes leading quickly to death. The strains could be recovered by supplementation with 33 μM ALA. Pathogenicity assays revealed the als1 strains were essentially non-pathogenic, inferring a key role for the synthesis of ALA during in planta growth. Supplementing the strains with ALA restored growth in vitro and also pathogenicity for up to 5 days after inoculation. Further examination by inoculating the als1 strains onto wounded leaves found that pathogenicity was only partially restored, suggesting that host-derived in planta levels of ALA are not sufficient to support growth. This study has identified a key role for fungal ALA synthesis during infection and revealed its potential as an antifungal target.

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Chang, C. C., C. C. McCormick, A. W. Lin, R. R. Dietert, and Y. J. Sung. "Inhibition of nitric oxide synthase gene expression in vivo and in vitro by repeated doses of endotoxin." American Journal of Physiology-Gastrointestinal and Liver Physiology 271, no. 4 (October 1, 1996): G539—G548. http://dx.doi.org/10.1152/ajpgi.1996.271.4.g539.

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We have examined the effects of repeated endotoxin administration in vivo and in vitro on the induction of nitric oxide synthase (NOS). In vivo, hepatic NOS activity and mRNA were increased markedly by the administration of Escherichia coli lipopolysaccharide (LPS). The change in hepatic NOS activity coincided with a marked accumulation of hepatic citrulline. Both enzyme activity and citrulline concentration returned to normal by 12 h after LPS administration. At this time, a subsequent administration of endotoxin caused no change in either NOS mRNA, NOS activity, or citrulline concentration, and thus an endotoxin-refractory state for nitric oxide (NO) synthesis was established. Normal sensitivity was reestablished by 24 h after the initial dose. In vitro studies using both a macrophage cell line (HD11) and primary macrophages indicated that LPS pretreatment caused cells in culture to become completely refractory to subsequent stimulation by LPS. Finally, we tested the hypothesis that NO may be involved in the development of the refractory state. Various inhibitors blocked the initial synthesis of NO by > 90% but failed to influence the development of the refractory state. Our study demonstrates both in vivo and in vitro that NO synthesis is completely blocked after repeated exposure to endotoxin by a mechanism that appears to be pretranslational. This model of early endotoxin tolerance may provide insight into the molecular mechanisms that regulate expression of the NOS gene.

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48

Stephens, Jennifer L., Soo Hee Lee, Kimberly S. Paul, and Paul T. Englund. "Mitochondrial Fatty Acid Synthesis in Trypanosoma brucei." Journal of Biological Chemistry 282, no. 7 (December 12, 2006): 4427–36. http://dx.doi.org/10.1074/jbc.m609037200.

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Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and β-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [14C]pyruvate or [14C]threonine, either of which is catabolized to [14C]acetyl-CoA in the mitochondrion. Although some of the [14C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.

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AMARA, Amro A., and Bernd H. A. REHM. "Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues." Biochemical Journal 374, no. 2 (September 1, 2003): 413–21. http://dx.doi.org/10.1042/bj20030431.

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The class II PHA (polyhydroxyalkanoate) synthases [PHAMCL synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHAMCLs using CoA thioesters of medium-chain-length 3-hydroxyfatty acids (C6–C14) as a substrate. Only recently PHAMCL synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHAMCL synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the α/β-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the α/β-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved γ-turns of the α/β-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the α/β-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

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Sato, Shun, Hiroshi Habe, Tokuma Fukuoka, Dai Kitamoto, and Keiji Sakaki. "Stepwise synthesis of 2,3-O-dipalmitoyl-D-glyceric acid and an in vitro evaluation of its cytotoxicity." Journal of Oleo Science 61, no. 6 (2012): 337–41. http://dx.doi.org/10.5650/jos.61.337.

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Journal articles on the topic 'Synthèss in vitro'

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