Biochemical and kinetic properties of quinone oxidoreductase from Leishmania orientalis, a member of medium-chain dehydrogenase/reductase superfamily

Abstract

Soluble quinone oxidoreductase (QOR) is an attractive target for treating leishmaniasis. In this study, a QOR from Leishmania orientalis PCM2 (LoQOR) was identified as a member of nonZn²⁺ medium-chain dehydrogenase/reductase (MDR) superfamily. Using steady-state and rapid kinetics approaches, it was found that LoQOR catalyzes the electron transfer from NADH to menadione via ping-pong kinetics which is unusual for this enzyme. The reaction mechanism was proposed accordingly. The modeled structures created by AlphaFold version 3.0 revealed crucial residues, i.e., Thr131, Tyr134, and Arg268, which may involve the binding to carboxamide group of the NADH nicotinamide ring. The kinetic analysis of the LoQOR mutants compared to the wild-type revealed that these residues are not essential for overall catalysis. However, the kinetic mechanism of Tyr134Phe, Tyr134His, and Arg268Gln mutants has shifted to the ternary complex (LoQOR:NADH:menadione) model, except for Thr131Val and Arg268Lys mutants. It is hypothesized that Tyr134 and Arg268 are likely responsible for determining the mode of the catalytic mechanism. Hence, our findings here provide a fundamental insight into the MDR-type QORs and other related non-metal enzymes in the MDR superfamily.