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Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17429 42667 2009 7 PDF Available
Title
Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis
Abstract

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.

Keywords
Phenol-oxidases; Rational mutagenesis; C-terminal tail; Enzyme stability
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Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 45, Issues 6–7, December 2009, Pages 507–513
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