Structural and Kinetic Studies on Native Intermediates and an Intermediate Analogue in Benzoylformate Decarboxylase Reveal a Least Motion Mechanism with an Unprecedented Short-Lived Predecarboxylation Intermediate

摘要

ABSTRACT: The thiamin diphosphate- (ThDP-) dependent enzyme benzoylformate decarboxylase (BFDC)ncatalyzes the nonoxidative decarboxylation of benzoylformic acid to benzaldehyde and carbon dioxide.nTo date, no structural information for a cofactor-bound reaction intermediate in BFDC is available. Fornkinetic analysis, a chromophoric substrate analogue was employed that produces various absorbingnintermediates during turnover but is a poor substrate with a 104-fold compromised kcat. Here, we havenanalyzed the steady-state distribution of native intermediates by a combined chemical quench/1H NMRnspectroscopic approach and estimated the net rate constants of elementary catalytic steps. At substratensaturation, carbonyl addition of the substrate to the cofactor (k′ ∼ 500 s-1 at 30 °C) and elimination ofnbenzaldehyde (k′ ∼ 2.400 s-1) were found to be partially rate-determining for catalysis, whereasndecarboxylation of the transient 2-mandelyl-ThDP intermediate is 1 order of magnitude faster with k′ ∼n16.000 s-1, the largest rate constant of decarboxylation in any thiamin enzyme characterized so far. ThenX-ray structure of a predecarboxylation intermediate analogue was determined to 1.6 Å after cocrystallizationnof BFDC from Pseudomonas putida with benzoylphosphonic acid methyl ester. In contrast to thenfree acid, for which irreversible phosphorylation of active center Ser26 was reported, the methyl esternforms a covalent adduct with ThDP with a similar configuration at C2R as observed for other thiaminnenzymes. The C2-C2R bond of the intermediate analogue is out of plane by 7°, indicating strain. Thenphosphonate part of the adduct forms hydrogen bonds with Ser26 and His281, and the 1-OH group isnheld in place by interactions with His70 and the 4′-amino group of ThDP. The phenyl ring accommodatesnin a hydrophobic pocket formed by Phe464, Phe397, Leu109, and Leu403. A comparison with thenpreviously determined structure of BFDC in noncovalent complex with the inhibitor (R)-mandelate suggestsna least motion mechanism. Binding of benzoylphosphonic acid methyl ester to BFDC was furtherncharacterized by CD spectroscopy and stopped-flow kinetics, indicating a two-step binding mechanismnwith a 200-fold slower carbonyl addition to ThDP than determined for benzoylformic acid, in line withnthe observed slight structural reorganization of Phe464 due to steric clashes with the phosphonate moiety.