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Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954

Lookup NU author(s): Professor Paul RaceORCiD

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Abstract

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied. © 2007 Elsevier Ltd. All rights reserved.

Publication metadata

Author(s): Race PR, Lovering AL, White SA, Grove JI, Searle PF, Wrighton CW, Hyde EI

Publication type: Article

Publication status: Published

Journal: Journal of Molecular Biology

Year: 2007

Volume: 368

Issue: 2

Pages: 481-492

Print publication date: 27/04/2007

Online publication date: 11/02/2007

ISSN (print): 0022-2836

Publisher: Academic Press

URL: https://doi.org/10.1016/j.jmb.2007.02.012

DOI: 10.1016/j.jmb.2007.02.012

PubMed id: 17350040

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