Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity: mechanistic implication for amide hydrolysis by serine hydrolases.
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 (k(cat)/K(m)) 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 (k(cat)). The increase in k(cat) 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.
Nakagawa
Yuichi
Y
Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan.
Hasegawa
Atsuko
A
Hiratake
Jun
J
Sakata
Kanzo
K
D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D019020:Directed Molecular Evolution / Q000379:methods*; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism
D008049:Lipase; D012697:Serine Endopeptidases; D000581:Amidohydrolases