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Structural basis of Escherichia coli nitroreductase NfsB triple mutants engineered for improved activity and regioselectivity toward the prodrug CB1954

Paper ID Volume ID Publish Year Pages File Format Full-Text
34274 45016 2015 7 PDF Available
Title
Structural basis of Escherichia coli nitroreductase NfsB triple mutants engineered for improved activity and regioselectivity toward the prodrug CB1954
Abstract

•The single mutant F124W regioselectively reduces the 4-NO2 group of CB1954.•Mutations F123A and T41L increase the kcat values in two different ways.•Two triple mutants were obtained with superior activity and regioselectivity.

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 kcat/Km 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 kcat values in two different ways, leading to improved enzyme activity.

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Keywords
Escherichia coli nitroreductase NfsB; CB1954; Protein engineering; X-ray structure
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Structural basis of Escherichia coli nitroreductase NfsB triple mutants engineered for improved activity and regioselectivity toward the prodrug CB1954
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 11, November 2015, Pages 1760–1766
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us