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 (pKa of 6.2) for TSA formation from pH 7.0-7.7 in wild type GCL. This pKa is similar to the pKa of 6.1 for the [1′,4′-iminopyrimidine (IP)]/[4′-aminopyrimidinium (APH+)] protonic equilibrium, suggesting that the same group(s) 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 acetyl donor and glyoxylate as acceptor, implying that this substitutent favored pyruvate as donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the pre-decarboxylation intermediate analogs 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.

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

“…In almost all such enzymes this depends on an active-site Glu residue, although in at least one case, glyoxalate carboligase, in which the Glu is replaced with a Val, this is achieved through other protein interactions (Kaplun et al, 2008;Nemeria et al, 2012). In Mtb-MenD, this role is assumed by Glu55, which has been shown by mutagenesis of Ec-MenD to be essential for activity (Bhasin et al, 2003).…”

Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…Addition of D-GAP increased the rate constant of decarboxylation by 600-fold. This is a particularly interesting case mechanistically, related to glyoxylase carboligase (GCL), where ThDP-catalyzed decarboxylation of the donor substrate is coupled to carboligation with the acceptor substrate [65].…”

“…This substitution is an attempt to recapitulate the role of the missing conserved glutamate in this enzyme [65].…”

“…Right. Structure of the active center of GCL demonstrating the hydrophobic environment of the thiazolium ring of ThDP (RCSB structure 2PAN)[65].…”

Using site-saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate-dependent enzymes

“…, a hydrogen bond to the ionized glutamate facilitates formation of the imino tautomer and, ultimately, the formation of the ylide necessary for enzyme activity . With the exception of glyoxylate carboligase , the glutamate is conserved throughout the family and its replacement reduces enzyme activity by more than four orders of magnitude .…”