Characterization of NfsB, the minor oxygen-insensitive nitroreductase of Escherichia coli.

摘要

Nitro-substituted compounds constitute a wide range of chemicals whose potent biological activity has significant human health implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase (NR) activities that can be classified on the basis of oxygen sensitivity. The recent accumulation of genomic data has helped lead to the recognition of the oxygen-insensitive NRs as a widespread family of enzymes that now includes an estimated 140 sequences in organisms throughout the three main domains of life. The physiological role of these enzymes remains a mystery. Escherichia coli has two oxygen-insensitive NRs, NfsA and NAB. NAB, the focus of this study, is a FMN-containing flavoprotein with a monomeric molecular mass of approximately 24 kDa, and can use either NADH or NADPH as a source of reducing equivalents.; The research reported in this thesis contributes to the characterization of E. coli NAB in four main ways. First, in a combined effort to elucidate the development of 5-nitrofuran resistance in E. coli , it was determined that the increased resistance associated with second step nitrofuran-resistant mutants is a consequence of mutational inactivation of the nfsB gene. An unusually high proportion of mutational events in nfsB involved IS elements. Second, it was demonstrated that NfsB is expressed in response to induction of the SoxRS and MarRAB regulons, key components of the E. coli global response to environmental stress. Third, an affinity purification scheme for NAB was developed and the protein was kinetically characterized. Detailed kinetic analysis using nitrofurazone as a substrate shows that NfsB has a catalytic specificity (kcat/Km value) of approximately 200 muM -1sec-1 for NADH and NADPH. Mechanistically, the kinetic data are consistent with the notion that NfsB uses a ping-pong bi-bi mechanism. Seventeen individual mutations were introduced by PCR site-directed mutagenesis. One mutation, G158S, resulted in a decreased specificity for NADPH, while another, F70S, increased the turnover number (kcat) for nitrofurazone without affecting cofactor specificity. Fourth, the active site of NfsB was characterized by computer analysis, leading to suggestions for protein pockets that could be targets for drug development.