Journal articles: 'Coenzyme enzyme complex'

1

Tsai, C. Stan, and D. J. Senior. "Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria." Biochemistry and Cell Biology 68, no. 4 (April 1, 1990): 751–57. http://dx.doi.org/10.1139/o90-108.

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Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD+- and NADP+-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.Key words: aldehyde dehydrogenase, coenzyme preference.

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Speranza, Giovanna, Wolfgang Buckel, and Bernard T. Golding. "CoenzymeB12-dependent enzymatic dehydration of 1,2-diols: simple reaction, complex mechanism!" Journal of Porphyrins and Phthalocyanines 08, no. 03 (March 2004): 290–300. http://dx.doi.org/10.1142/s1088424604000271.

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The conversion of glycerol to acrolein is an undesirable event in whisky production, caused by infection of the broth with Klebsiella pneumoniae. This organism uses glycerol dehydratase to transform glycerol into 3-hydroxypropanal, which affords acrolein on distillation. The enzyme requires adenosylcobalamin (coenzyme B12) as cofactor and a monovalent cation (e.g. K+). Diol dehydratase is a similar enzyme that converts 1,2-diols ( C2- C4) including glycerol into an aldehyde and water. The subtle stereochemical features of these enzymes are exemplified by propane-1,2-diol: both enantiomers are substrates but different hydrogen and oxygen atoms are abstracted. The mechanism of action of the dehydratases has been elucidated by protein crystallography and ab initio molecular orbital calculations, aided by stereochemical and model studies. The 5'-deoxyadenosyl (adenosyl) radical from homolysis of the coenzyme's Co - C σ-bond abstracts a specific hydrogen atom from C -1 of diol substrate giving a substrate radical that rearranges to a product radical by 1,2-shift of hydroxyl from C -2 to C -1. The rearrangement mechanism involves an acid-base 'push-pull' in which migration of OH is facilitated by partial protonation by Hisα143, synergistically assisted by partial deprotonation of the non-migrating ( C -1) OH by the carboxylate of Gluα170. The active site K+ion holds the two hydroxyl groups in the correct conformation, whilst not significantly contributing to catalysis. Recently, diol dehydratases not dependent on coenzyme B12have been discovered. These enzymes utilize the same kind of diol radical chemistry as the coenzyme B12-dependent enzymes and they also use the adenosyl radical as initiator, but this is generated from S-adenosylmethionine.

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Ehebauer, Matthias, Madhan Anandhakrishnan, Michael Zimmermann, Elke Noens, Arjen Jakobi, Carsten Sachse, Uwe Sauer, and Matthias Wilmanns. "AccD1 And AccA1 from M. tuberculosis form A dodecameric MCC-type holo complex." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C429. http://dx.doi.org/10.1107/s2053273314095709.

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Mycobacteria have an unusual redundancy of six putative carboxyltransferase genes that form high-molecular weight holo acyl coenzyme A carboxylase complexes with a complementary set of three biotin carboxylase genes. Most of these enzyme complexes use small fatty acid coenzyme A esters as substrate, to allow their extension by one methylene group via a carboxybiotin-mediated α-carboxylation reaction. Redundant occurrence of these complexes was assumed to be related to highly complex enzymatic requirements in lipid biosynthesis, as the mycobacterial thick cell wall comprises unusual very long chain fatty acids, including mycolic acid. We have solved two high-resolution crystal structures of the 350 kDa hexameric assemblies of two different acyl coenzyme A carboxylase hexameric assemblies, AccD5 and AccD6 [1; Anandhakrishnan et al., unpublished], and characterized these enzyme complexes functionally. In a second step we investigated the acyl coenzyme A carboxylase complex AccD1-AccA1 from Mycobacteria tuberculosis with hitherto unknown function. By using a metabolomics approach we found that AccD1-AccA1 is involved in branched amino acid catabolism, which was not investigated in mycobacteria before [Ehebauer et al, unpublished. Using an in vitro assay, we show that the enzyme complex uses methylcrotonyl coenzyme A as substrate]. We determined the overall architecture of the 700 kDa AccD1-AccA1 complex to be formed from three layers of a central AccD1 hexameric ring, flanked by two distal tiers composed of three AccA1 subunits each. Our electron microscopy data match the overall dimensions of a methylcrotonyl coenzyme A holo complex with known structure and thus support our functional findings. Our data suggest a unique functional role of the AccD1-AccA1 complex within the Mycobacterium tuberculosis acyl coenzyme A carboxylase interactome. Ultimately, it is our goal to solve this and related structures of ACCase holo complexes by high-resolution crystallography as well. The abstract is dedicated to Louis Delbaere with whom I shared time during my PhD at the University of Basel, Switzerland.

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Teixido, Francisco, Dolores De Arriaga, Félix Busto, and Joaquin Soler. "Cytoplasmic malate dehydrogenase from Phycomyces blakesleeanus: Kinetics and mechanism." Canadian Journal of Biochemistry and Cell Biology 63, no. 10 (October 1, 1985): 1097–105. http://dx.doi.org/10.1139/o85-137.

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The kinetics and reaction mechanism of cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) in 0.1 M potassium phosphate buffer (pH 7.5) at 30 °C have been investigated. The initial rate and product inhibition studies were consistent with an ordered bi-bi mechanism that involved more than one kinetically significant ternary complex and also with the coenzyme binding first. The dissociation of the coenzyme from the enzyme–coenzyme complex appeared to be the slowest step in either direction of the reaction. The kinetic and rate constants for the individual steps of the reaction were determined.

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Kang, Lin-Woo, Sandra B. Gabelli, Mario A. Bianchet, Wen Lian Xu, Maurice J. Bessman, and L. Mario Amzel. "Structure of a Coenzyme A Pyrophosphatase from Deinococcus radiodurans: a Member of the Nudix Family." Journal of Bacteriology 185, no. 14 (July 15, 2003): 4110–18. http://dx.doi.org/10.1128/jb.185.14.4110-4118.2003.

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ABSTRACT Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg2+, its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

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Bell, E. T., C. LiMuti, C. L. Renz, and J. E. Bell. "Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes." Biochemical Journal 225, no. 1 (January 1, 1985): 209–17. http://dx.doi.org/10.1042/bj2250209.

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The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

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7

Friedmann, Silke, Astrid Steindorf, Birgit E. Alber, and Georg Fuchs. "Properties of Succinyl-Coenzyme A:l-Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus." Journal of Bacteriology 188, no. 7 (April 1, 2006): 2646–55. http://dx.doi.org/10.1128/jb.188.7.2646-2655.2006.

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ABSTRACT The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by l-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:l-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for l-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:l-malate CoA transferase forms a large (αβ)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + l-malate → succinate + l-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts l-citramalate instead of l-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.

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Leskovac, Vladimir, Svetlana Trivic, and Draginja Pericin. "Isomerization of an enzyme-coenzyme complex in yeast alcohol dehydrogenase-catalysed reactions." Journal of the Serbian Chemical Society 68, no. 2 (2003): 77–84. http://dx.doi.org/10.2298/jsc0302077l.

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In this work, all the rate constants in the kinetic mechanism of the yeast alcohol dehydrogenase-catalyzed oxidation of ethanol by NAD+, at pH 7.0, 25 ?C, have been estimated. The determination of the individual rate constants was achieved by fitting the reaction progress curves to the experimental data, using the procedures of the FITSIM and KINSIM software package of Carl Frieden. This work is the first report in the literature showing the internal equilibrium constants for the isomerization of the enzyme-NAD+ complex in yeast alcohol dehydrogenase-catalyzed reactions.

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9

Winberg, J. O., and J. S. McKinley-McKee. "Drosophila melanogaster alcohol dehydrogenase: product-inhibition studies." Biochemical Journal 301, no. 3 (August 1, 1994): 901–9. http://dx.doi.org/10.1042/bj3010901.

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The Drosophila melanogaster alleloenzymes AdhS and AdhF have been studied with respect to product inhibition by using the two substrate couples propan-2-ol/acetone and ethanol/acetaldehyde together with the coenzyme couple NAD+/NADH. With both substrate couples the reaction was consistent with an ordered Bi Bi mechanism. The substrates added to the enzyme in a compulsory order, with coenzyme as the leading substrate, to give two interconverting ternary complexes. The second ternary complex broke down with release of products in an obligatory order, with the aldehyde/ketone leaving first. Both the acetaldehyde and acetone products formed binary complexes with the enzyme that affected NAD+ binding. However, only an enzyme-acetone complex seemed to affect NADH binding and hence the reverse reaction. The inhibitory pattern with acetaldehyde as product was also affected by the formation of a ternary enzyme-NAD(+)-acetaldehyde complex, which broke down to acetic acid and NADH. The product-inhibition pattern shown in the present work is different from that published for Drosophila Adh previously and this discrepancy can not be explained by the use of different variants of Drosophila Adh.

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10

Stiborová, Marie, and Sylva Leblová. "Mechanism of action of heavy metals, S-triazine herbicides and nitrates on alcohol dehydrogenase from rape." Collection of Czechoslovak Chemical Communications 51, no. 8 (1986): 1781–88. http://dx.doi.org/10.1135/cccc19861781.

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Heavy metals (Pb2+, Cu2+, Cd2+, and Zn2+) inhibited alcohol dehydrogenase (ADH) of rape (EC 1.1.1.1.); Pb2+ and Cd2+ ions affected the imidazole ring of histidine, Cu2+ and Zn2+ ions interacted with the sulphydryl groups of cysteine in the molecule of the enzyme. The coenzyme protected ADH from inactivation by Pb2+ and Cd2+, but did not protect it from Cu2+ and Zn2+. Ethanol in a ternary complex ADH-NAD+ -ethanol was a strong protecting agent from Pb2+ and Cd2+ ions. Nitrates inhibited rape ADH toward any substrate. Sulphates and fluorides had no effect. The S-triazine herbicides studied proved strong inhibitors of rape ADH (their Ki's corresponded to concentrations of the order 10-4 mol 1-1), occupying the binding site for the coenzyme. The herbicides interacted with the metallic component of the enzyme, to which the nicotinamide part of the coenzyme is bound.

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Griffin, Joanna, and Paul C. Engel. "The –SH Protection Method for Determining Accurate Kd Values for Enzyme-Coenzyme Complexes of NAD+-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes." Enzyme Research 2010 (June 29, 2010): 1–5. http://dx.doi.org/10.4061/2010/951472.

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Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of Kd for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD+, NADH and NADPH at various concentrations in protecting against inactivation by 200 μM DTNB allowed determination of Kd values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the Kd determinations indicated that, although Kd values for NAD+ and NADH were increased considerably, Kd for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.

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Grishin, Andrey M., Eunice Ajamian, Linhua Zhang, and Miroslaw Cygler. "Crystallization and preliminary X-ray analysis of PaaAC, the main component of the hydroxylase of theEscherichia coliphenylacetyl-coenzyme A oxygenase complex." Acta Crystallographica Section F Structural Biology and Crystallization Communications 66, no. 9 (August 26, 2010): 1045–49. http://dx.doi.org/10.1107/s174430911002748x.

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TheEscherichia coli paaoperon encodes enzymes of the phenylacetic acid-utilization pathway that metabolizes phenylacetate in the form of a coenzyme A (CoA) derivative. The phenylacetyl-coenzyme A oxygenase complex, which has been postulated to contain five components designated PaaABCDE, catalyzes ring hydroxylation of phenylacetyl-CoA. The PaaAC subcomplex shows low sequence similarity to other bacterial multicomponent monooxygenases (BMMs) and forms a separate branch on the phylogenetic tree. PaaAC, which catalyzes the hydroxylation reaction, was purified and crystallized in the absence of a bound ligand as well as in complexes with CoA, 3-hydroxybutyryl-CoA, benzoyl-CoA and the true substrate phenylacetyl-CoA. Crystals of the ligand-free enzyme belonged to space groupP212121and diffracted to 2.65 Å resolution, whereas complexes with CoA and its derivatives crystallized in space groupP41212 and diffracted to ∼2.0 Å resolution. PaaAC represents the first crystallized BMM hydroxylase that utilizes a CoA-linked substrate.

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Hidalgo-Gutiérrez, Agustín, Pilar González-García, María Elena Díaz-Casado, Eliana Barriocanal-Casado, Sergio López-Herrador, Catarina M. Quinzii, and Luis C. López. "Metabolic Targets of Coenzyme Q10 in Mitochondria." Antioxidants 10, no. 4 (March 26, 2021): 520. http://dx.doi.org/10.3390/antiox10040520.

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Coenzyme Q10 (CoQ10) is classically viewed as an important endogenous antioxidant and key component of the mitochondrial respiratory chain. For this second function, CoQ molecules seem to be dynamically segmented in a pool attached and engulfed by the super-complexes I + III, and a free pool available for complex II or any other mitochondrial enzyme that uses CoQ as a cofactor. This CoQ-free pool is, therefore, used by enzymes that link the mitochondrial respiratory chain to other pathways, such as the pyrimidine de novo biosynthesis, fatty acid β-oxidation and amino acid catabolism, glycine metabolism, proline, glyoxylate and arginine metabolism, and sulfide oxidation metabolism. Some of these mitochondrial pathways are also connected to metabolic pathways in other compartments of the cell and, consequently, CoQ could indirectly modulate metabolic pathways located outside the mitochondria. Thus, we review the most relevant findings in all these metabolic functions of CoQ and their relations with the pathomechanisms of some metabolic diseases, highlighting some future perspectives and potential therapeutic implications.

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Zhang, Yanli, Linley R. Schofield, Carrie Sang, Debjit Dey, and Ron S. Ronimus. "Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9." Archaea 2017 (2017): 1–6. http://dx.doi.org/10.1155/2017/5793620.

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(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

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Kuchmenko, E. B., D. N. Petukhov, G. V. Donchenko, L. S. Mkhitaryan, S. V. Tymoshchuk, N. A. Strutynskaya, G. L. Vavilova, and V. F. Sagach. "Effect of complexes of precursors and modulators of coenzyme q biosynthesis on functional state of old rats' heart mitochondria." Biomeditsinskaya Khimiya 56, no. 2 (2010): 244–56. http://dx.doi.org/10.18097/pbmc20105602244.

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Our research demonstrate that ageing leads to changes in activity of electron-transporting enzyme complexes in myocardial mitochondria of old rats and to increased sensitivity of mitochondrial permeability transition pore to inductors of its opening - Ca2+ and phenylarsine oxide. We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) we observed the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensivity of lipid and protein free-radical peroxidation in the heart and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be used to treat mitochondrial dysfunction under numerous pathologies of cardiovascular system, as well as in ageing.

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Shima, Seigo, Gangfeng Huang, Tristan Wagner, and Ulrich Ermler. "Structural Basis of Hydrogenotrophic Methanogenesis." Annual Review of Microbiology 74, no. 1 (September 8, 2020): 713–33. http://dx.doi.org/10.1146/annurev-micro-011720-122807.

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Most methanogenic archaea use the rudimentary hydrogenotrophic pathway—from CO2 and H2 to methane—as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420–based hydride transfers, and one coenzyme F430–based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

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NEUHAUSER, Wilfried, Dietmar HALTRICH, Klaus D. KULBE, and Bernd NIDETZKY. "NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme." Biochemical Journal 326, no. 3 (September 15, 1997): 683–92. http://dx.doi.org/10.1042/bj3260683.

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During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.

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Meikle, P. J., A. M. Whittle, and J. J. Hopwood. "Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation." Biochemical Journal 308, no. 1 (May 15, 1995): 327–33. http://dx.doi.org/10.1042/bj3080327.

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Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

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Al-Kassim, Loola S., and C. Stan Tsai. "Purification and kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity." Biochemistry and Cell Biology 71, no. 9-10 (September 1, 1993): 421–26. http://dx.doi.org/10.1139/o93-062.

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A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)+-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.Key words: purification, pickerel liver, alcohol dehydrogenase.

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García-Muriana, Francisco J., María C. Alvarez-Ossorioa, María M. Sánchez-Garcés, F. de la Rosa, and Angel M. Relimpio. "Further Characterization of Aspartate Aminotransferase from Haloferax mediterranei: Pyridoxal Phosphate as Coenzyme and Inhibitor." Zeitschrift für Naturforschung C 50, no. 3-4 (April 1, 1995): 241–47. http://dx.doi.org/10.1515/znc-1995-3-413.

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The enzyme aspartate aminotransferase has been isolated from the halophilic bacterium Haloferax mediterranei in its apoenzyme form. The interaction with its coenzyme (pyridoxal phosphate) has been investigated. For concentrations up to 0.05 mᴍ, the incubation with pyridoxal phosphate reconstituted the active complex (holoenzyme) following a second order kinetic with a k2 of 5.2 min-1mᴍ-1. This active complex showed a dissociation constant (Kd) of 7.8 x 10-6 ᴍ. For concentrations higher than 0.1 mᴍ, pyridoxal phosphate produced an inactivation process with a complex second order kinetic. This inactivation is partially reverted by dialysis or by lysine treatment. Thus, after 80% of inactivation, 55% of the original activity is recovered by a long-time dialysis, and with 50 mᴍ lysine also a partial reactivation (among 20-33%) is observed. The enzyme treated with 1 mᴍ pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the modified enzyme presents a smaller size due to a conformational change.

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Smith, Clyde A., Marta Toth, Thomas M. Weiss, Hilary Frase, and Sergei B. Vakulenko. "Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia revealed by crystallographic and small-angle X-ray scattering analysis." Acta Crystallographica Section D Biological Crystallography 70, no. 10 (September 27, 2014): 2754–64. http://dx.doi.org/10.1107/s1399004714017635.

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Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6′)-Ie-APH(2′′)-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6′)-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2′′)-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2′′)-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6′)-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6′)-Ie enzyme is joined to APH(2′′)-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2′′)-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6′)-Ie-APH(2′′)-Ia enzyme.

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Scott, A. Ian, and Charles A. Roessner. "Recent discoveries in the pathways to cobalamin (coenzyme B12) achieved through chemistry and biology." Pure and Applied Chemistry 79, no. 12 (January 1, 2007): 2179–88. http://dx.doi.org/10.1351/pac200779122179.

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The genetic engineering of Escherichia coli for the over-expression of enzymes of the aerobic and anaerobic pathways to cobalamin has resulted in the in vivo and in vitro biosynthesis of new intermediates and other products that were isolated and characterized using a combination of bioorganic chemistry and high-resolution NMR. Analyses of these products were used to deduct the functions of the enzymes that catalyze their synthesis. CobZ, another enzyme for the synthesis of precorrin-3B of the aerobic pathway, has recently been described, as has been BluB, the enzyme responsible for the oxygen-dependent biosynthesis of dimethylbenzimidazole. In the anaerobic pathway, functions have recently been experimentally confirmed for or assigned to the CbiMNOQ cobalt transport complex, CbiA (a,c side chain amidation), CbiD (C-1 methylation), CbiF (C-11 methylation), CbiG (lactone opening, deacylation), CbiP (b,d,e,g side chain amidation), and CbiT (C-15 methylation, C-12 side chain decarboxylation). The dephosphorylation of adenosylcobalamin-phosphate, catalyzed by CobC, has been proposed as the final step in the biosynthesis of adenosylcobalamin.

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23

Cassetta, Alberto, Ivet Krastanova, Katja Kristan, Mojca Brunskole Švegelj, Doriano Lamba, Tea Lanišnik Rižner, and Jure Stojan. "Insights into subtle conformational differences in the substrate-binding loop of fungal 17β-hydroxysteroid dehydrogenase: a combined structural and kinetic approach." Biochemical Journal 441, no. 1 (December 14, 2011): 151–60. http://dx.doi.org/10.1042/bj20110567.

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The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8 μM; at 100 μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π–π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme–coenzyme–steroid ternary complexes.

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24

Wang, Shuning, Haiyan Huang, Johanna Moll, and Rudolf K. Thauer. "NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri." Journal of Bacteriology 192, no. 19 (July 30, 2010): 5115–23. http://dx.doi.org/10.1128/jb.00612-10.

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ABSTRACT It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred 2− + NADH + 2 NADP+ + H+ = Fdox + NAD+ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.

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25

Chase, T. "Mannitol-1-phosphate dehydrogenase of Escherichia coli Chemical properties and binding of substrates." Biochemical Journal 239, no. 2 (October 15, 1986): 435–43. http://dx.doi.org/10.1042/bj2390435.

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Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5′-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5′-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.

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26

Makukh, Y. M., T. M. Gryvul, A. Ya Krasnevich, and D. S. Vignan. "Aldosereductase: structure, mechanism of action, biological role and functioning according to normo and hyperglycemia." Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies 22, no. 99 (October 28, 2020): 125–42. http://dx.doi.org/10.32718/nvlvet9920.

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The review summarizes the literature on the structure, biological role and mechanism of action of aldose reductase at different blood glucose levels. Aldosereductase is the first enzyme of the sorbitol (polyol) pathway to glucose metabolism. In mammals, it is a monomeric protein with a molecular weight of 32–56 kDa, has 347–370 amino acid remainders. Its secondary structure consists of α-helices and β-bends, which alternate in 8 units. The active site of the enzyme is located at the C-terminus of the β-bend and contains a glutathione-binding domain. The active site of aldose reductase consists of two sites: substrate-binding and catalytic. The first is formed mainly by the residues of hydrophilic amino acids, and the second, by hydrophobic ones. The interaction of the enzyme with a coenzyme causes conformational changes in aldose reductase. It is believed that the enzyme functions according to the principle of an ordered “bi-bi” mechanism, that is, the coenzyme binds first, and the oxidized product is released last. The reduction of aldehydes of aldose reductase includes several stages: the interaction of the enzyme with NADPH and the formation of a binary complex, the acceptance of the substrate and the formation of a ternary complex (enzyme-coenzyme-substrate) and the separation of the alcohol-reaction product and the oxidized coenzyme. According to normoglycemia in mammalian cells via the sorbitol pathway, up to 1–3 % of intracellular glucose is restored. Under these conditions, it reduces the content of toxic and reactive aldehydes such as: 4-hydroxy-trans-2-nonenal, malondialdehyde, glyoxal, acrolein and their conjugates with reduced glutathione and carnosine, which are also toxic. Before being excreted from the body, they are reduced by aldose reductase to non-toxic compounds. Thus, the enzyme is one of the components of the body's antioxidant system. Hyperglycemia, which is most pronounced in diabetes, significantly increases the flow of glucose through the sorbitol pathway. The activation of aldosereductase and sorbitol dehydrogenase causes the use of a significant amount of NADPH, which leads to a decrease in antioxidant protection, and the excessive formation of NADH leads to a violation of the ratio of reduced and oxidized forms, known as “pseudohypoxia”. Metabolites of the sorbitol pathway, which are formed in excessive amounts, get toxic effects on metabolism and cellular structures, in particular: sorbitol, as an osmotically active component, causes lens edema, leads to the formation of cataracts, and fructose, fructose-phosphate and 3-deoxyglucasone underlie the pathogenesis of secondary diabetic complications.

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Lawrence, Sarah H., and James G. Ferry. "Steady-State Kinetic Analysis of Phosphotransacetylase from Methanosarcina thermophila." Journal of Bacteriology 188, no. 3 (February 1, 2006): 1155–58. http://dx.doi.org/10.1128/jb.188.3.1155-1158.2006.

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ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.

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28

Gago, Gabriela, Daniel Kurth, Lautaro Diacovich, Shiou-Chuan Tsai, and Hugo Gramajo. "Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from Mycobacterium tuberculosis." Journal of Bacteriology 188, no. 2 (January 15, 2006): 477–86. http://dx.doi.org/10.1128/jb.188.2.477-486.2006.

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ABSTRACT Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the α biotinylated subunit AccA3, the carboxyltransferase β subunit AccD5, and the ε subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The α and β subunits are capable of forming a stable α6-β6 subcomplex but with very low specific activity. The addition of the ε subunit, which binds tightly to the α-β subcomplex, is essential for gaining maximal enzyme activity.

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Kumar, Neeraj, Shubin Liu, and Pawel M. Kozlowski. "Charge Separation Propensity of the Coenzyme B12–Tyrosine Complex in Adenosylcobalamin-Dependent Methylmalonyl–CoA Mutase Enzyme." Journal of Physical Chemistry Letters 3, no. 8 (April 9, 2012): 1035–38. http://dx.doi.org/10.1021/jz300102s.

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30

Ryle, C. M., and K. F. Tipton. "Kinetic studies with the low-Km aldehyde reductase from ox brain." Biochemical Journal 227, no. 2 (April 15, 1985): 621–27. http://dx.doi.org/10.1042/bj2270621.

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Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

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31

Johnson, Celeste L. V., Marian L. Buszko, and Thomas A. Bobik. "Purification and Initial Characterization of the Salmonella enterica PduO ATP:Cob(I)alamin Adenosyltransferase." Journal of Bacteriology 186, no. 23 (December 1, 2004): 7881–87. http://dx.doi.org/10.1128/jb.186.23.7881-7887.2004.

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ABSTRACT The PduO enzyme of Salmonella enterica is an ATP:cob(I)alamin adenosyltransferase that catalyzes the final step in the conversion of vitamin B12 to coenzyme B12. The primary physiological role of this enzyme is to support coenzyme B12-dependent 1,2-propanediol degradation, and bioinformatic analysis has indicated that it has two domains. Here the PduO adenosyltransferase was produced in Escherichia coli, solubilized from inclusion bodies, purified to apparent homogeneity, and partially characterized biochemically. The Km values of PduO for ATP and cob(I)alamin were 19.8 and 4.5 μM, respectively, and the enzyme V max was 243 nmol min−1 mg of protein−1. Further investigations showed that PduO was active with ATP and partially active with deoxy-ATP, but lacked measurable activity with other nucleotides. 31P nuclear magnetic resonance established that triphosphate was a product of the PduO reaction, and kinetic studies indicated a ternary complex mechanism. A series of truncated versions of the PduO protein were produced in Escherichia coli, partially purified, and used to show that adenosyltransferase activity is associated with the N-terminal domain. The N-terminal domain was purified to near homogeneity and shown to have biochemical properties and kinetic constants similar to those of the full-length enzyme. This indicated that the C-terminal domain was not directly involved in catalysis or substrate binding and may have another role.

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32

Barthová, Jana, Irena Hulová, and Miroslava Birčáková. "Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean." Collection of Czechoslovak Chemical Communications 59, no. 2 (1994): 467–72. http://dx.doi.org/10.1135/cccc19940467.

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The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.

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33

PETSCHACHER, Barbara, Stefan LEITGEB, Kathryn L. KAVANAGH, David K. WILSON, and Bernd NIDETZKY. "The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography." Biochemical Journal 385, no. 1 (December 14, 2004): 75–83. http://dx.doi.org/10.1042/bj20040363.

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CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

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Tjhin, Erick T., Vanessa M. Howieson, Christina Spry, Giel G. van Dooren, and Kevin J. Saliba. "A novel heteromeric pantothenate kinase complex in apicomplexan parasites." PLOS Pathogens 17, no. 7 (July 29, 2021): e1009797. http://dx.doi.org/10.1371/journal.ppat.1009797.

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Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.

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35

Miller, Tarryn E., Thomas Beneyton, Thomas Schwander, Christoph Diehl, Mathias Girault, Richard McLean, Tanguy Chotel, et al. "Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts." Science 368, no. 6491 (May 7, 2020): 649–54. http://dx.doi.org/10.1126/science.aaz6802.

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Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chemical energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compositions and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biological worlds.

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36

Brinsmade, Shaun R., Tenzin Paldon, and Jorge C. Escalante-Semerena. "Minimal Functions and Physiological Conditions Required for Growth of Salmonella enterica on Ethanolamine in the Absence of the Metabolosome." Journal of Bacteriology 187, no. 23 (December 1, 2005): 8039–46. http://dx.doi.org/10.1128/jb.187.23.8039-8046.2005.

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ABSTRACT During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO2. In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme B12 are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

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Keep, N. H., G. A. Smith, M. C. W. Evans, G. P. Diakun, and P. F. Leadlay. "The synthetic substrate succinyl(carbadethia)-CoA generates cob(II)alamin on adenosylcobalamin-dependent methylmalonyl-CoA mutase." Biochemical Journal 295, no. 2 (October 15, 1993): 387–92. http://dx.doi.org/10.1042/bj2950387.

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Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent methylmalonyl-CoA mutase, has been prepared by a simplified procedure. When recombinant mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-methylmalonyl-CoA mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.

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Lowe, D. M., and P. K. Tubbs. "3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative." Biochemical Journal 227, no. 2 (April 15, 1985): 601–7. http://dx.doi.org/10.1042/bj2270601.

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Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a ‘half-of-the-sites’ reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.

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Plapp, Bryce V., David C. Sogin, Robert T. Dworschack, David P. Bohlken, Christoph Woenckhaus, and Reinhard Jeck. "Kinetics and native and modified liver alcohol dehydrogenase with coenzyme analogs: isomerization of enzyme-nicotinamide adenine dinucleotide complex." Biochemistry 25, no. 19 (September 23, 1986): 5396–402. http://dx.doi.org/10.1021/bi00367a008.

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Leutwein, Christina, and Johann Heider. "Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria." Journal of Bacteriology 183, no. 14 (July 15, 2001): 4288–95. http://dx.doi.org/10.1128/jb.183.14.4288-4295.2001.

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ABSTRACT Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-inducedbbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates.

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Bartucci, Roberta, Anna Salvati, Peter Olinga, and Ykelien L. Boersma. "Vanin 1: Its Physiological Function and Role in Diseases." International Journal of Molecular Sciences 20, no. 16 (August 9, 2019): 3891. http://dx.doi.org/10.3390/ijms20163891.

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The enzyme vascular non-inflammatory molecule-1 (vanin 1) is highly expressed at gene and protein level in many organs, such as the liver, intestine, and kidney. Its major function is related to its pantetheinase activity; vanin 1 breaks down pantetheine in cysteamine and pantothenic acid, a precursor of coenzyme A. Indeed, its physiological role seems strictly related to coenzyme A metabolism, lipid metabolism, and energy production. In recent years, many studies have elucidated the role of vanin 1 under physiological conditions in relation to oxidative stress and inflammation. Vanin’s enzymatic activity was found to be of key importance in certain diseases, either for its protective effect or as a sensitizer, depending on the diseased organ. In this review, we discuss the role of vanin 1 in the liver, kidney, intestine, and lung under physiological as well as pathophysiological conditions. Thus, we provide a more complete understanding and overview of its complex function and contribution to some specific pathologies.

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42

Timofeev, Vladimir, Evgenia Smirnova, Larisa Chupova, Roman Esipov, and Inna Kuranova. "X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase fromMycobacterium tuberculosisduring the catalyzed reaction." Acta Crystallographica Section D Biological Crystallography 68, no. 12 (November 9, 2012): 1660–70. http://dx.doi.org/10.1107/s0907444912040206.

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Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) fromMycobacterium tuberculosis(PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Å resolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt–ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on Cαatoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme–substrate and enzyme–product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

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González, Javier M., Ricardo Marti-Arbona, Julian C. H. Chen, Brian Broom-Peltz, and Clifford J. Unkefer. "Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase." Acta Crystallographica Section F Structural Biology Communications 74, no. 10 (September 19, 2018): 610–16. http://dx.doi.org/10.1107/s2053230x18011809.

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Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

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Song, Haigang, Hoi Pang Sung, Yuk Sing Tse, Ming Jiang, and Zhihong Guo. "Ligand-dependent active-site closure revealed in the crystal structure ofMycobacterium tuberculosisMenB complexed with product analogues." Acta Crystallographica Section D Biological Crystallography 70, no. 11 (October 23, 2014): 2959–69. http://dx.doi.org/10.1107/s1399004714019440.

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1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase catalyzes an essential intramolecular Claisen condensation in menaquinone biosynthesis and is an important target for the development of new antibiotics. This enzyme inMycobacterium tuberculosisis cofactor-free and is classified as a type II DHNA-CoA synthase, differing from type I enzymes, which rely on exogenous bicarbonate for catalysis. Its crystal structures in complex with product analogues have been determined at high resolution to reveal ligand-dependent structural changes, which include the ordering of a 27-residue active-site loop (amino acids 107–133) and the reorientation of the carboxy-terminal helix (amino acids 289–301) that forms part of the active site from the opposing subunit across the trimer–trimer interface. These structural changes result in closure of the active site to the bulk solution, which is likely to take place through an induced-fit mechanism, similar to that observed for type I DHNA-CoA synthases. These findings demonstrate that the ligand-dependent conformational changes are a conserved feature of all DHNA-CoA synthases, providing new insights into the catalytic mechanism of this essential tubercular enzyme.

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Jun, A. S., I. A. Trounce, M. D. Brown, J. M. Shoffner, and D. C. Wallace. "Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia." Molecular and Cellular Biology 16, no. 3 (March 1996): 771–77. http://dx.doi.org/10.1128/mcb.16.3.771.

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A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

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Skursk, Ladislav, Miroslav Řezáĉ, Allah Nawaz Khan, Lukáŝ Žídek, and Jaroslav Roĉek. "Hydroperoxide Inhibitor of Horse Liver Alcohol Dehydrogenase Activity, Tightly Bound to the Enzyme-Nad+Complex, Characteristically Degrades the Coenzyme." Journal of Enzyme Inhibition 6, no. 3 (January 1992): 211–22. http://dx.doi.org/10.3109/14756369209020171.

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Keinanen, T. A., T. Hyvonen, J. Vepsalainen, L. Alhonen, A. R. Khomutov, and J. Janne. "Stable analogues of coenzyme-substrate complex of spermidine/spermine-N 1-acetyltransferase reaction. Synthesis and interaction with the enzyme." Russian Journal of Bioorganic Chemistry 40, no. 2 (March 2014): 155–61. http://dx.doi.org/10.1134/s1068162014020071.

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Bohren, K. M., J. P. von Wartburg, and B. Wermuth. "Kinetics of carbonyl reductase from human brain." Biochemical Journal 244, no. 1 (May 15, 1987): 165–71. http://dx.doi.org/10.1042/bj2440165.

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Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.

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Kirilenko, Bogdan M., Lee R. Hagey, Stephen Barnes, Charles N. Falany, and Michael Hiller. "Evolutionary Analysis of Bile Acid-Conjugating Enzymes Reveals a Complex Duplication and Reciprocal Loss History." Genome Biology and Evolution 11, no. 11 (October 31, 2019): 3256–68. http://dx.doi.org/10.1093/gbe/evz238.

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Abstract To fulfill their physiological functions, bile acids are conjugated with amino acids. In humans, conjugation is catalyzed by bile acid coenzyme A: amino acid N-acyltransferase (BAAT), an enzyme with a highly conserved catalytic triad in its active site. Interestingly, the conjugated amino acids are highly variable among mammals, with some species conjugating bile acids with both glycine and taurine, whereas others conjugate only taurine. The genetic origin of these bile acid conjugation differences is unknown. Here, we tested whether mutations in BAAT’s catalytic triad could explain bile acid conjugation differences. Our comparative analysis of 118 mammals first revealed that the ancestor of placental mammals and marsupials possessed two genes, BAAT and BAATP1, that arose by a tandem duplication. This duplication was followed by numerous gene losses, including BAATP1 in humans. Losses of either BAAT or BAATP1 largely happened in a reciprocal fashion, suggesting that a single conjugating enzyme is generally sufficient for mammals. In intact BAAT and BAATP1 genes, we observed multiple changes in the catalytic triad between Cys and Ser residues. Surprisingly, although mutagenesis experiments with the human enzyme have shown that replacing Cys for Ser greatly diminishes the glycine-conjugating ability, across mammals we found that this residue provides little power in predicting the experimentally measured amino acids that are conjugated with bile acids. This suggests that the mechanism of BAAT’s enzymatic function is incompletely understood, despite relying on a classic catalytic triad. More generally, our evolutionary analysis indicates that results of mutagenesis experiments may not easily be extrapolatable to other species.

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Wang, Lei, and K. M. J. Menon. "Regulation of Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Expression in the Rat Ovary: Relationship to Cholesterol Metabolism." Endocrinology 146, no. 1 (January 1, 2005): 423–31. http://dx.doi.org/10.1210/en.2004-0805.

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Down-regulation of LH/human chorionic gonadotropin (hCG) receptor (LHR) mRNA in the ovary after the preovulatory LH surge or the administration of a pharmacological dose of LH/hCG occurs through a posttranscriptional mechanism. A LHR mRNA-binding protein was identified as the LHR mRNA destabilizing factor, and its identity was established as mevalonate kinase (Mvk). In the present study, we determined that, in the pseudopregnant rat ovary, LHR mRNA levels began to fall 4 h after hCG injection, at which time Mvk protein levels were elevated, and this elevation was preceded by an increase in Mvk mRNA levels. When the cytosolic fractions of hCG-treated ovaries were subjected to RNA EMSA, an increase in LHR mRNA-LHR mRNA-binding protein complex formation was observed, in parallel with the increase of Mvk expression. We also found that hCG coordinately up-regulated the expression of Mvk and other sterol-responsive elements containing cholesterol biosynthesis enzymes, such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and farnesyl pyrophosphate synthase. This up-regulation was transient, but the hCG-induced ovarian cholesterol depletion lasted for more than 24 h. Taken together, our results suggest that, in the ovary, LH/hCG up-regulates the expression of cholesterol biosynthesis enzymes and lipoprotein receptors to replenish cellular cholesterol, and the up-regulation of Mvk leads to a down-regulation of LHR and suppresses the LH/hCG signal cascade transiently. Thus Mvk, an enzyme involved in cholesterol biosynthesis, serves as a link between LHR mRNA expression and cellular cholesterol metabolism.

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Journal articles on the topic 'Coenzyme enzyme complex'

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