The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I: Purification and properties of a nitroreductase enzyme from Escherichia coli—A potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)

Abstract

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the “classical nitroreductase” of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active against CB1954 ( k cat=360 min −) han Walker DT diaphorase ( K cat = 4 min −1) and also has a lower K m for NADH (6 vs 75 μM).