Enzymes from the NfsB subgroup are commonly referred to as “classical nitroreductases”, and catalyze the reduction of a broad range of substrates including a diverse range of nitroaromatics, quinones, flavins, and metals. Activity, albeit low, has also been observed with enones.
Bryant, D. W., McCalla, D. R., Leeksma, M. & Laneuville, P.
Type I nitroreductases of Escherichia coli.
▸ Abstract
Analysis of partially purified crude extract of Escherichia coli K12 by chromatography and gel electrophoresis has resulted in the separation of three distinct activities which catalyse the reduction of nitrofurazone (semicarbazone of 5-nitro-2-furaldehyde) in the presence of oxygen (type I nitroreductases). The major enzymatic activity (type IA), which was dependent solely on NADPH as a cofactor, was absent from nitrofurazone-resistant strains NFR 402 and NFR 502, but present in SIL 41, a strain which is only marginally resistant to the nitrofuran. The remaining nitroreductase activities (IB1 and IB2) utilize either NADH or NADPH as a cofactor. These activities coelute from DEAE-cellulose at pH 7.2, but may be differentiated by their behaviour on CM-cellulose at pH 5.8. The reductase activity missing in SIL 41 was observed in extracts of strain NFR 402 but not NFR 502. This enzyme (IB1) though retained by DEAE-cellulose had no affinity for CM-cellulose. The only reductase present in extracts of NFR 502 (a nitrofuran-resistant strain selected after two mutational events) was type IB2. This activity, also detectable in SIL 41 and NFR 402, has not been mapped genetically. An interesting feature of the type IB2 enzyme is its apparent inactivation by MnCl2 which has been routinely used as a partial purification step in the past.
Can. J. Microbiol.
1981;27(None):81-86
| PubMed ID:
7011517
Zenno, S., Koike, H., Tanokura, M. & Saigo, K.
Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri.
▸ Abstract
nfsB, encoding a minor oxygen-insensitive nitroreductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major flavin reductase from Vibrio fischeri and classical nitroreductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-flavin oxidoreductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.
J. Biochem.
1996;120(None):736-744
| PubMed ID:
8947835
Watanabe, M., Nishino, T., Takio, K., Sofuni, T. & Nohmi, T.
Purification and characterization of wild-type and mutant ‘classical’ nitroreductases of Salmonella typhimurium. L33R mutation greatly diminishes binding of FMN to the nitroreductase of S. typhimurium
▸ Abstract
"Classical" nitroreductase of Salmonella typhimurium is a flavoprotein that catalyzes the reduction of nitroaromatics to metabolites that are toxic, mutagenic, or carcinogenic. This enzyme represents a new class of flavin-dependent enzymes, which includes nitroreductases of Enterobacter cloacae and Escherichia coli, flavin oxidoreductase of Vibrio fischeri, and NADH oxidase of Thermus thermophilus. To investigate the structure-function relation of this class of enzymes, the gene encoding a mutant nitroreductase was cloned from S. typhimurium strain TA1538NR, and the enzymatic properties were compared with those of the wild-type. DNA sequence analysis revealed a T to G mutation in the mutant nitroreductase gene, predicting a replacement of leucine 33 with arginine. In contrast to the wild-type enzyme, the purified protein with a mutation of leucine 33 to arginine has no detectable nitroreductase activities in the standard assay conditions and easily lost FMN by dialysis or ultrafiltration. In the presence of an excess amount of FMN, however, the mutant protein exhibited a weak but measurable enzyme activity, and the substrate specificity was similar to that of the wild-type enzyme. Possible mechanisms by which the mutation greatly diminishes binding of FMN to the nitroreductase are discussed.
J. Biol. Chem.
1998;273(None):23922-23928
| PubMed ID:
9727006
Roldán, M. D., Pérez-Reinado, E., Castillo, F. & Moreno-Vivián, C.
Reduction of polynitroaromatic compounds: the bacterial nitroreductases.
▸ Abstract
Most nitroaromatic compounds are toxic and mutagenic for living organisms, but some microorganisms have developed oxidative or reductive pathways to degrade or transform these compounds. Reductive pathways are based either on the reduction of the aromatic ring by hydride additions or on the reduction of the nitro groups to hydroxylamino and/or amino derivatives. Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds. Nitroreductases have raised a great interest due to their potential applications in bioremediation, biocatalysis, and biomedicine, especially in prodrug activation for chemotherapeutic cancer treatments. Different bacterial nitroreductases have been purified and their biochemical and kinetic parameters have been determined. The crystal structure of some nitroreductases have also been solved. However, the physiological role(s) of these enzymes remains unclear. Nitroreductase genes are widely spread within bacterial genomes, but are also found in archaea and some eukaryotic species. Although studies on regulation of nitroreductase gene expression are scarce, it seems that nitroreductase genes may be controlled by the MarRA and SoxRS regulatory systems that are involved in responses to several antibiotics and environmental chemical hazards and to specific oxidative stress conditions. This review covers the microbial distribution, types, biochemical properties, structure and regulation of the bacterial nitroreductases. The possible physiological functions and the biotechnological applications of these enzymes are also discussed.
FEMS Microbiol Rev
2008;32(None):474-500
| PubMed ID:
18355273
Yanto, Y., Hall, M. & Bommarius, A. S.
Nitroreductase from Salmonella typhimurium: characterization and catalytic activity.
▸ Abstract
The biocatalytic activity of nitroreductase from Salmonella typhimurium (NRSal) was investigated for the reduction of alpha,beta-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics. The synthesized gene was subcloned into a pET28 overexpression system in E.coli BL21 strain, and the corresponding expressed protein was purified to homogeneity with 15% protein mass yield and 41% of total activity recovery. NRSal showed broad substrate acceptance for various nitro compounds such as 1-nitrocyclohexene and aliphatic nitroalkenes (alkene reductase activity), as well as nitrobenzene (nitroreductase activity), with substrate conversion efficiency of > 95%. However, the reduction of enones was generally low, proceeding albeit with high stereoselectivity. The efficient biocatalytic reduction of substituted nitroalkenes provides a route for the preparation of the corresponding nitroalkanes. NRSal also demonstrated the first single isolated enzyme-catalyzed reduction of nitrobenzene to aniline through the formation of nitrosobenzene and phenylhydroxylamine as intermediates. However, chemical condensation of the two intermediates to produce azoxybenzene currently limits the yield of aniline.
Org. Biomol. Chem.
2010;8(8):1826-1832
| PubMed ID:
20449486
