We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.
The crystal structure of the thiamin diphosphate (ThDP)-dependent enzyme benzoylformate decarboxylase (BFD), the third enzyme in the mandelate pathway of Pseudomonas putida, has been solved by multiple isomorphous replacement at 1.6 A resolution and refined to an R-factor of 15.0% (free R = 18.6%). The structure of BFD has been compared to that of other ThDP-dependent enzymes, including pyruvate decarboxylase. The overall architecture of BFD resembles that of the other family members, and cofactor- and metal-binding residues are well conserved. Surprisingly, there is no conservation of active-site residues not directly bound to the cofactor. The position of functional groups in the active site may be conserved, however. Three classes of metal ions have been identified in the BFD crystal structure: Ca2+ bound to the cofactor in each subunit, Mg2+ on a 2-fold axis of the tetramer, and Ca2+ at a crystal contact. The structure includes a non-proline cis-peptide bond and an unusually long and regular polyproline type II helix that mediates the main contact between tetramers in the crystal. The high-quality electron-density map allowed the correction of errors totaling more than 10% of the amino acid sequence, which had been predicted from the reported sequence of the mdlC gene. Analysis of the BFD structure suggests that requirements for activation of the cofactor, the nature of the reaction intermediates, and architectural considerations relating to the protein fold have been dominant forces in the evolution of ThDP-dependent enzymes.
Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.
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