A second dihydroorotate dehydrogenase (type A) of the human pathogen Enterococcus faecalis: Expression, purification, and steady-state kinetic mechanism

We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococc us faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of L-dihy droorotate while reducing a number of substrates, including fumarate, coenz yme Q(0), and menadione. The steady-state kinetic mechanism has been determ ined with menadione as an oxidizing substrate at pH 7.5. Initial velocity a nd product inhibition data suggest that the enzyme follows a two-site noncl assical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dih ydroorotate. In contrast, titration of the enzyme with menadione had no eff ect on FMN absorbance, consistent with nonoverlapping binding sites for dih yroorotate and menadione, as suggested from the kinetic mechanism. The redu ctive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data in dicate that the oxidative half-reaction is a rate-limiting segment, while o rotate, most likely, retains significant affinity for the reduced enzyme, a s suggested by the product inhibition pattern. (C) 2000 Academic Press.