Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde.
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.
Hibbert
Edward G
EG
Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
Senussi
Tarik
T
Smith
Mark E B
ME
Costelloe
Sean J
SJ
Ward
John M
JM
Hailes
Helen C
HC
Dalby
Paul A
PA
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence / Q000502:physiology; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism*
D000447:Aldehydes; C005556:propionaldehyde; D014174:Transketolase; D000079:Acetaldehyde; C010972:glycolaldehyde