Novel TdsD nitroreductase: characterization of kinetics and substrate specificity.

Abstract

The reduction of quinones and nitroaromatic compounds catalyzed by Type I nitroreductases is important due to its role in their potential cytotoxic effects and/or biodegradation. The main goal of this work was to investigate the mechanism of catalysis of a TdsD nitroreductase (NR) (TdsD1), a member from an understudied branch of the nitroreductase superfamily, derived from a soil metagenome study. Like the Type I NRs NfsA and NfsB, TdsD1 performed two-electron reduction of quinones and four-electron reduction of nitroaromatic compounds according to a "ping-pong" mechanism with a rate-limiting oxidative half-reaction. TdsD1 was also inhibited by the classical inhibitors of other NRs, dicoumarol and Cibacron blue. Despite sharing only a low degree of homology with the NfsA and NfsB subfamily enzymes, sequence comparisons and computer modelling point to the possibility of an analogous FMN isoalloxazine ring location within the intersubunit space of TdsD1. It also possesses similar specificity for nitroaromatic compounds and quinones, in particular the shared characteristic of being especially active with 2-hydroxy-1,4-naphthoquinone derivatives. It is possible that the similar character of binding of oxidants and other ligands relative to the NfsA and NfsB subfamily enzymes may be related to the conserved Arg27 and Ser53 residues in the active site of TdsD1.