Structural and Kinetic Analysis of Catalysis by a Thiamin Diphosphate-Dependent Enzyme, Benzoylformate Decarboxylase

Abstract

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.

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense

“…As both enzymes are part of a pathway for the syn- Several ThDP-dependent 2-keto acid decarboxylases such as pyruvate decarboxylase from Saccharomyces cerevisiae (ScPDC) and from Kluyveromyces lactis are activated by their substrate (15,17,33). Most bacterial pyruvate decarboxylases, IPDC Ec , and benzoylformate decarboxylase display hyperbolic Michaelis-Menten kinetics without any indication of substrate activation (11,27,30). The combination of clear substrate activation for certain substrates and hyperbolic MichaelisMenten kinetics (or only very weak substrate activation) for others in a single enzyme, as observed for the PPDC Ab , is less common.…”

“…In the benzoylformate decarboxylase of Pseudomonas putida, the glutamate residue is also replaced by Leu. Here, protonation of the enamine-carbanion intermediate is proposed to be mediated by a catalytic histidine residue in the active site (27). In PPDC Ab , it was proposed that the leucine, which is located underneath the carboxylate group of the lactyl-ThDP intermediate, increases the hydrophobic character of the active site and consequently stabilizes the zwitterionic intermediates (40).…”

Glutamate 636 of the Escherichia coli Pyruvate Dehydrogenase-E1 Participates in Active Center Communication and Behaves as an Engineered Acetolactate Synthase with Unusual Stereoselectivity

“…Also, as with YPDC, it appears that it is the negative charge on these residues which helps to avoid the side reaction by electrostatic repulsion vis à vis the second molecule of pyruvate, because with both YPDC and PDHc-E1, conversion of the aspartate (YPDC) or glutamate (PDHc-E1) to the amidated form or to alanine led to the same outcome. It is interesting to speculate why on benzoylformate decarboxylase, an enzyme analogous to YPDC, where the methyl group of pyruvate is replaced by a phenyl ring, the residue Ser 26 occupies the position corresponding to Asp 28 of YPDC (30,31). Perhaps, although the YPDC and PDHc-E1 require the negative charge at Asp 28 and Glu 636 , respectively, to repel the second pyruvate, on BFD the likelihood of this side reaction is diminished because of the much larger size of the substrate benzoylformate compared with pyruvate.…”

Identification of Novel Benzoylformate Decarboxylases by Growth Selection

“…4.1.1.7) catalyzes the formation of benzaldehyde from benzoylformate by decarboxylation. The structure of BFD was solved in the absence (11) and the presence (34) of mandelic acid as an inhibitor, confirming that the enzyme acts as a tetramer, and active-site residues have been elucidated by site-directed mutants (34,40) and directedevolution studies (26,27).…”

“…3). (34,40), and the boxed residues are involved in thiamine diphosphate cofactor and metal binding.…”