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Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone: Reversed binding orientations in different redox states of the enzyme

Lookup NU author(s): Professor Paul RaceORCiD


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The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ∼10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Publication metadata

Author(s): Race PR, Lovering AL, Green RM, Ossor A, White SA, Searle PF, Wrighton CJ, Hyde EI

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2005

Volume: 280

Issue: 14

Pages: 13256-13264

Print publication date: 08/04/2005

Online publication date: 31/01/2005

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: Elsevier Inc.


DOI: 10.1074/jbc.M409652200

PubMed id: 15684426


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