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Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Paper ID Volume ID Publish Year Pages File Format Full-Text
24773 43536 2008 6 PDF Available
Title
Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde
Abstract

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.

Keywords
DSW, deep square well; Ery, erythrulose; E4P, erythrose-4-phosphate; GA, glycolaldehyde; HPA, hydroxypyruvate; DHP, 1,3-dihydroxypentan-2-one; TK, transketolase; TPP, thiamine pyrophosphate; TPTC, 2,3,5-triphenyltetrazolium chlorideDirected evolution; Bio
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Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 134, Issues 3–4, 30 April 2008, Pages 240–245
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