Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

Abstract

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg 2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

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

“…The initial identification of the IP form (positive CD band, 300-314 nm) resulted from formation of a stable pre-decarboxylation adduct of ThDP with (a) MAP or acetylphosphinate [43,44] (CH 3 C (@O)P(H)O 2 Na, AcP - [43]), with pyruvate-specific enzymes and (b) the aromatic 2-oxo acid analogue methyl benzoylphosphonate (MBP) with BFDC and BAL [26,67]. With 12 enzymes tested so far, the IP form appeared on the stopped-flow time scale (either absorption or CD mode): the reaction is efficiently catalyzed by all of the enzymes.…”

“…The enzyme benzaldehyde lyase (BAL, EC 4.1.2.38) carries out reversible decomposition of (R)-benzoin to two molecules of benzaldehyde; in the reverse direction the enzyme is a carboligase. The X-ray structure of BAL contained only two acid-base residues surrounding the ThDP at the active center [24][25][26]: a highly conserved Glu50 within hydrogen bonding distance of the N1 0 atom of the 4 0 -aminopyrimidine ring and a His29 residue. The residue His29 is too far from the thiazolium C2 atom to be of value in the first steps of the reaction and was suggested to have a function in removing the b-hydroxyl proton of the ThDP-bound benzoin to assist in releasing the first benzaldehyde molecule.…”

“…1). From the large number of high-resolution X-ray structures available for more than 20 years, starting with the structures of transketolase [29] (TK), pyruvate oxidase [30] (POX) from Lactobacillus plantarum and pyruvate decarboxylase from the yeast Saccharomyces cerevisae (YPDC) [31,32], it has become clear that the negative charges with the required Mg 2+ or Ca 2+ , the divalent metal serving as an anchor in a highly tailored environment with a universally conserved GDG recognition site and the diphosphateMg 2+ binding motif consisting of GDG-X- 26 -NN sequence of amino acids suggested by the Perham group [33]. As shown in a series of seminal papers by Breslow, the thiazolium ring is central to catalysis [34], due to its ability to form a key nucleophilic center the C2 atom, the C2-carbanion/ylide or carbene -depending on one's viewpoint regarding the relative importance of the resonance contributions.…”

Catalyzing decarboxylation by taming carbon dioxide

“…Benzaldehyde lyase is a ThDP-dependent enzyme that is not a decarboxylase (Scheme 6) [56,57]. The enzyme becomes an active decarboxylase with benzoylformate as a substrate when serine is in place of an alanine residue at the active site [58].…”

“…Scheme 6: A proposed route for inactivation by a phosphonate and catalysis of decarboxylation by a serine side chain based on the inactivation of benzoyformate decarboxyalse by a phosphonate analogue of benzyolformate[56,57].…”