1887

Abstract

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from . The encoding gene was identified as a homologue of the of , and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the and genes; however, the and genes of were not separated by a fourth gene, . The gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor ( 3 μM) and activity with this substrate was inhibited by the presence of dicoumarol ( 10 μM). In contrast, YwrO showed a marked preference for NADPH as a cofactor ( 40 μM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. YwrO was shown to be a flavoprotein with a monomeric molecular mass of 215 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties YwrO more closely resembles DTD than NTR. Its for CB 1954 was lower than that of NTR (617 μM compared to 862 μM). Enhanced cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and YwrO. The work has led to the identification of a previously unknown nitroreductase, YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

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2002-01-01
2024-03-29
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