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PEDS Advance Access published online on July 6, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm025
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity: mechanistic implication for amide hydrolysis by serine hydrolases

Yuichi Nakagawa, Atsuko Hasegawa, Jun Hiratake1 and Kanzo Sakata

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

1 To whom correspondence should be addressed. E-mail: hiratake{at}scl.kyoto-u.ac.jp

A lipase from Pseudomonas aeruginosa was subjected to directed evolution for increased amidase activity to probe the catalytic mechanism of serine hydrolases for the hydrolysis of amides. Random mutagenesis combined with saturation mutagenesis for all the amino acid residues at the substrate-binding site successfully identified the mutation at the residue 252 next to the catalytic H251 as a hot spot for selectively increasing the amidase activity of the lipase. The saturation mutagenesis targeted for the oxyanion hole (M16 and H83) gave no positive results. The substitutions of Met or Phe for Leu252 significantly increased the amidase activity toward N-(2-naphthyl)oleamide (2), whereas the esterase activity toward structurally similar 2-naphthyl oleate (1) was not affected by the substitution. The triple mutant F207S/A213D/M252F (Sat252) exhibited amidase activity (kcat/Km) 28-fold higher than that of the wild-type lipase. Kinetic analysis of Sat252 and its parental clone 10F12 revealed that the amidase activity was increased by the increase in the catalytic efficiency (kcat). The increase in kcat suggested the importance of the leaving group protonation by the catalytic His during the break down of the tetrahedral intermediate in the hydrolysis of amides.

Keywords: amidase activity/catalytic triad/directed evolution/leaving group protonation/Pseudomonas aeruginosa lipase

Received February 12, 2007; revised May 12, 2007; accepted May 14, 2007.


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