The accidental assignment of function in the tautomerase superfamily

Abstract

Cg10062 from Corynebacterium glutamicum is a tautomerase superfamily member with the characteristic β Àα À β fold and catalytic Pro-1. It is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue with high sequence similarity (53%) that includes the six critical active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114). However, Cg10062 is a poor cis-CaaD: it has much lower catalytic efficiency and lacks isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene (2,3-butadienote) were investigated as potential substrates for Cg10062. Cg10062 is a hydratase/decarboxylase using propiolate and cis-3-chloro-and 3-bromoacrylates, where malonate semialdehyde is the product of hydration and acetaldehyde is the product of decarboxylation. The two activities occur consecutively using the initial substrate. In contrast, 2-butynoate and 2,3-butadienote only undergo a hydration reaction with Cg10062 to afford acetoacetate. cis-CaaD does not function as a hydratase/decarboxylase using any of these substrates, yielding only the products of hydration. Cg10062 proceeds by direct hydration or covalent catalysis (using Pro-1) depending on the substrate. Direct hydration yields the hydration products and covalent catalysis yields the hydration and decarboxylation products. Cg10062 mutants shift the reaction toward one or the other mechanism. The observation that propiolate is the best substrate suggests that Cg10062 could be a hydratase/decarboxylase in a pathway that transforms an unknown acetylene compound to acetaldehyde via propiolate. The bifunctional activity of Cg10062 might also have implications for the evolution of the dehalogenase and decarboxylase activities in the tautomerase superfamily.

Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation

“…11,21 Compound 10 is not processed to any appreciable extent. 28 For both of the substrates, the hydration product (4 or 11, respectively) is not decarboxylated by the enzyme.…”

Tetralone derivatives are MIF tautomerase inhibitors and attenuate macrophage activation and amplify the hypothermic response in endotoxemic mice

“…In the reversible docking models, the position of the electrophilic methylene bridge relative to Pro1A hints at the possibility of a covalent mechanism. Such catalytic proline residues reportedly possess the ability to mediate enzymatic conversions through covalent catalysis 66 . NAPQI has been shown to bind covalently to Pro1, while exhibiting non-covalent inhibitory activity 67 .…”

Cg10062 Catalysis Forges a Link between Acetylenecarboxylic Acid and Bacterial Metabolism

“…We directed our focus onto Cg10062 due to its catalytic superiority with ACA relative to cis- CaaD . Additionally, active site mutants of Cg10062 that lacked hydratase/decarboxylase activity were previously identified by Whitman .…”

“…We directed our focus onto Cg10062 due to its catalytic superiority with ACA relative to cis-CaaD. 43 Additionally, active site mutants of Cg10062 that lacked hydratase/ decarboxylase activity were previously identified by Whitman. 44 Incubation of variants E114Q and E114D with ACA, followed by analysis of the resulting products using 1 H NMR indicated that these variants possessed hydratase activity without hydratase/decarboxylase activity.…”

“…Unfortunately, our efforts to re-isolate this organism and enzyme were unsuccessful. We therefore shifted our focus to cis- CaaD and Cg10062, two enzymes extensively described by Whitman and co-workers, which also convert ACA into MSA. − cis -CaaD-catalyzed hydration of ACA produces MSA exclusively, albeit at a slow rate. However, Cg10062 catalyzes hydration and hydration-dependent decarboxylation to produce a mixture of MSA and acetaldehyde (Figure ).…”