title: 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.
authors.affiliation: Department of Life Sciences,  Ben-Gurion University , P.O. Box 653, Beer-Sheva 84105, Israel.
authors.forename: Natalia
authors.initials: N
authors.lastname: Nemeria
authors.forename: Elad
authors.initials: E
authors.lastname: Binshtein
authors.forename: Hetalben
authors.initials: H
authors.lastname: Patel
authors.forename: Anand
authors.initials: A
authors.lastname: Balakrishnan
authors.forename: Ilan
authors.initials: I
authors.lastname: Vered
authors.forename: Boaz
authors.initials: B
authors.lastname: Shaanan
authors.forename: Ze'ev
authors.initials: Z
authors.lastname: Barak
authors.forename: David
authors.initials: D
authors.lastname: Chipman
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011743:Pyrimidines / Q000737:chemistry*; D019289:Pyruvic Acid; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011743:Pyrimidines; D018698:Glutamic Acid; D019289:Pyruvic Acid; D008025:Ligases; D013835:Thiamine Pyrophosphate