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Indexed by:Journal Papers

Date of Publication:2015-11-01

Journal:PROCESS BIOCHEMISTRY

Included Journals:SCIE、EI、Scopus

Volume:50

Issue:11

Page Number:1760-1766

ISSN No.:1359-5113

Key Words:Escherichia coli nitroreductase NfsB; CB1954; Protein engineering; X-ray structure

Abstract:Escherichia coli nitroreductase NfsB coupled with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] has great anti-cancer potential. However, its efficacy is limited by the low catalytic efficiency of NfsB against CB1954. In this paper, we show that substitution with a tryptophan at residue 124 strongly increases the selectivity toward the 4-NO2 group of CB1954, generating the more cytotoxic 4-hydroxylamine product. To further improve the activity, the F124W mutation and three previously reported beneficial mutations were combined randomly into double and triple mutants. Steady-state kinetic studies showed that they all exhibited enhanced activity toward CB1954. Two triple mutants, T41L/N71S/F124W and F123A/N71S/F124W, show a 9.2-17.2-fold increase in k(cat)/K-m compared with the wild-type and selectively reduce the 4-NO2 group of CB1954. The crystal structure of F123A/N71S/F124W was resolved. Comparison with the reported NfsB structures revealed that the F124W mutation may provide a stronger hydrophobic interaction with the aziridinyl group of CB1954, which was in favor of the reduction of the 4-NO2 group; while the F123A and T41L mutations increased the k(cat) values in two different ways, leading to improved enzyme activity. (C) 2015 Elsevier Ltd. All rights reserved.