A Transient Intermediate in the Reaction Catalyzed by (S)-Mandelate Dehydrogenase from Pseudomonas putida

Abstract

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

“…4) also suggested that the reverse reaction from pyruvate was not significant under the conditions used, which is consistent with the literature [21,25]. (Note that microscopic reversibility of a-hydroxy acid/a-keto acid interconversion is not called into question and was clearly shown for reactions catalyzed by mandelate dehydrogenase [45] and flavocytochrome b 5 [46]. However, oxidation of flavocytochrome b 5 by pyruvate occurred with a rate constant of only 0.08 s À1 ).…”

High Resolution Structures of an Oxidized and Reduced Flavoprotein

“…Affinity of Reduced MDH for the Product, BenzoylformateUnder anaerobic conditions, MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced enzyme by benzoylformate (27). The kinetic constants for the reverse reaction were determined by measuring increases in absorbance at 460 nm at 20°C for different benzoylformate concentrations.…”

Chemical Constituents and Antimicrobial Activities of Essential Oils Of syzygiumpolyanthum and Syzygiumaromaticum

“…It has been proven possessing antidiarrheal, antimicrobial, antihypertensive, and antioxidant activities in in-vitro studies. [9][10][11][12] S. aromaticum (clove, cengkih in Bahasa Indonesia) is indigenous to Indonesia and recently grown in India, Tanzania, Sri Lanka, Brazil, and Madagascar. It is used as an expectorant and to cure upset stomach.…”