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Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954

Centre for Applied Microbiology and Research (CAMR), Porton Down, Salisbury, Wiltshire SP4 0JG, UK¹

Author for correspondence: Gill M. Anlezark. Tel: +44 1980 612330. Fax: +44 1980 611310. e-mail: gill.anlezark{at}camr.org.uk

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, 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 B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K_m 3 µM) and activity with this substrate was inhibited by the presence of dicoumarol (K_i 1·0 µM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K_m 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. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21·5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K_m for CB 1954 was lower than that of NTR (617 µM compared to 862 µM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

Keywords: prodrug activation, DEPT, cancer therapy, flavoprotein, in vitro cytotoxicity

Abbreviations: ADEPT, antibody-directed enzyme prodrug therapy; CB 1954, 5-aziridinyl-2,4-dinitrobenzamide; DEPT, directed enzyme prodrug therapy; DTD, DT-diaphorase; GDEPT, gene-directed enzyme prodrug therapy; NTR, nitroreductase; SN 23862, bischloroethyl-2,4-dinitrobenzamide; VDEPT, viral-directed enzyme prodrug therapy; 2HX, 5-(aziridin-1-yl)-2-hydroxylamino-4-nitrobenzamide; 4HX, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide

The GenBank accession number for the sequence reported in this paper is AF373598.

^a Present address: Department of Biology, University of York, PO Box No. 373, York YO10 5YW, UK.

^b Present address: Nycomed-Amersham, Cell Based Assay Department, Cardiff Laboratories, Forest Farm, Whitchurch, Cardiff CF4 7YT, UK.

^c Present address: Perotsystems, 1 Oakwood Court, Sherwood Park, Nottingham NG15 0DR, UK.