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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.
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.
URL: https://doi.org/10.1074/jbc.M409652200
DOI: 10.1074/jbc.M409652200
PubMed id: 15684426
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