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Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

ABSTRACT: Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK(a) is similar to the pK(a) of 6.1 for the 1',4'-iminopyrimidine (IP)-4'-aminopyrimidinium (APH(+)) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH(+) form is accessible.

SUBMITTER: Nemeria N

PROVIDER: S-EPMC3493109 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

Nemeria Natalia N Binshtein Elad E Patel Hetalben H Balakrishnan Anand A Vered Ilan I Shaanan Boaz B Barak Ze'ev Z Chipman David D Jordan Frank F

Biochemistry 20120925 40

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7. ...[more]

PMID: 22970650

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Project description:The cocrystals of sulfamethazine with different acids, namely, 2-mercaptophenylcarboxylic acid, 2,6-pyridinedicarboxylic acid, 4-(4-hydroxyphenylazo)phenylcarboxylic acid, 3-(4-hydroxyphenyl)propanoic acid, and 4-(phenyl)phenylcarboxylic acid, are studied here. Each has distinct notable supramolecular features. The pyrimidin-2-amine unit of the sulfamethazine provided unique examples of cocrystals in which amidine and imidine forms or neutral and protonated forms of sulfamethazine are observed in 2:2 ratios. Hence, this study provides avenues to explore cocrystals with tautomeric forms together in a cocrystal and also neutral and protonated cocrystal partners as apparent multicomponents in cocrystals. Among the cocrystals, three of them have the amidine form of the sulfamethazine in respective self-assembly. The cocrystal of 2-mercapto-phenylcarboxylic acid with sulfamethazine has the amidine form and it has the distinction of having S-H···π interactions. The cocrystal of sulfamethazine with 2,6-pyridinecarboxylic acid is a rare example of a 1:1 cocrystal of sulfmethazine with dicarboxylic acid. It has methanol molecules as a solvent of crystallization. Sulfamethazine forms a hydrated cocrystal with 4-(4-hydroxyphenylazo)-phenylcarboxylic acid that has conventional R 2 2(8) synthons of amidine hydrogen-bonding with carboxylic acid. The phenolic part of the acid component is anchored to the water molecule and provides a robust self-assembly. The hydrated cocrystal of sulfamethazine with 3-(4-hydroxyphenyl)propanoic acid (2:2 cocrystal) has two independent molecules of sulfamethazine, one in amidine form and the other in imidine form. It has two neutral carboxylic acids anchored through complementary hydrogen bonds and also has two water molecules of crystallization. The cocrystal of sulfamethazine with 4-(phenyl)phenylcarboxylic acid is also a 2:2 cocrystal. It is a di-hydrate, which has a neutral and protonated form of sulfamethazine. The neutral form is hydrogen-bonded to a neutral carboxylic acid, whereas the protonated form is charge-assisted hydrogen-bonded to the corresponding carboxylate anion.

Dataset Information

Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

Publications

Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

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