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Structure–activity relationship of a cold-adapted purine nucleoside phosphorylase by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17319 42659 2012 7 PDF Available
Title
Structure–activity relationship of a cold-adapted purine nucleoside phosphorylase by site-directed mutagenesis
Abstract

Purine nucleoside phosphorylase can be expressed in Escherichia coli and the intact cells can be used as a catalyst for the biosynthesis of nucleosides. The purine nucleoside phosphorylases from E. coli (EcPNP) and Pseudoalteromonas sp. XM2107 (PsPNP) have been purified. In order to improve the catalytic efficiency, the model of three-dimensional structure of PsPNP was constructed, and then 9 active/binding-site mutants were constructed by one-step site-directed mutagenesis and characterized by steady-state kinetics. Double mutations exhibited the largest change of catalytic activity. The T90R:T156S mutant revealed 1000 fold enhancements in kcat/Km for inosine phosphorolysis. However, the T90A:T156A mutant revealed 500 fold reduction in catalytic activity when compared with wild-type one. These results in combination with the predicted locations of Thr90 and Thr156 side chains by homology modeling suggested that: (i) a complete hydrophobic pocket played an important role in the catalytic function of PsPNP; (ii) a potential transition state structure was present in hydrogen bond between the carboxyl groups of Thr90 in the phosphate binding site. Therefore, the application of site-directed mutagenesis will be benefit to further improve catalytic efficiency of PsPNP during the enzymatic synthesis of antivirus drug ribavirin.

► The 3D model of PsPNP was successfully constructed. ► The Val206, Thr156 and Thr90 of PsPNP are proved to be key sites. ► To further verify the catalytic activity by using enzymatic production of ribavirin.

Keywords
Site-directed mutagenesis; Purine nucleoside phosphorylase; Protein structure homology modeling; Enzymatic synthesis; Ribavirin
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Structure–activity relationship of a cold-adapted purine nucleoside phosphorylase by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issue 1, 10 June 2012, Pages 59–65
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