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

Enzyme and Microbial Technology
Xixian XieNing Chen

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 k(cat)/K(m) 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 furth...Continue Reading

References

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