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.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

“…We have identified two explanations regarding why the initial purification and cloning of an enzyme capable of CB1954 activation from E. coli isolated NfsB rather than NfsA . First, the assay used to monitor purification used the cofactor NADH, whereas NfsA has a strong preference for NADPH (see Figure 2) (Bryant et al, 1981;Zenno et al, 1996); furthermore, this assay used menadione as a substrate, which is reduced about twice as efficiently by NfsB as by NfsA (Zenno et al, 1996). Although the initial demonstration that NfsB could activate CB1954 (and does so B90-fold faster than rat DT-diaphorase ) led to the adoption of NfsB for prodrug activation gene therapy with CB1954, this paper presents evidence based on cell cultures that it may be advantageous to use NfsA in preference.…”

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

“…In the present work we have cloned and overexpressed the previously unknown product of a gene which was identified by sequence homology to be an orthologue of the ywrO of B. subtilis. In Bacillus spp., and amongst members of other genera (Bryant et al, 1981 ;Kinouchi & Ohnishi, 1983 ;Rosenkranz et al, 1982 ;G. M. Anlezark, unpublished data), total nitroreductase activity assayed in crude cell extracts often represents activity of at least three enzymes which can be purified individually only with difficulty (G. M. Anlezark, unpublished data).…”

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

“…Type I is oxygen insensitive (3,6,15,17,20,37,38,39), and type II is oxygen sensitive (21, 26). In type I, there are two nitroflavin reductase superfamiles: NfsA and NfsB (3,6,14,17,20,37,38,39). NfsA includes the major nitroreductase in E. coli (3, 37) and Frp in V. harveyi (14,17).…”

“…In type I, there are two nitroflavin reductase superfamiles: NfsA and NfsB (3,6,14,17,20,37,38,39). NfsA includes the major nitroreductase in E. coli (3,37) and Frp in V. harveyi (14,17). NfsB includes the minor nitroreductase in E. coli (3,20,38) and Frase I in Vibrio (Photobacterium) fischeri (6,39).…”

“…NfsA includes the major nitroreductase in E. coli (3,37) and Frp in V. harveyi (14,17). NfsB includes the minor nitroreductase in E. coli (3,20,38) and Frase I in Vibrio (Photobacterium) fischeri (6,39).…”