Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

Shosuke Yoshida, Junichi Enoki, Robert Kourist, Kenji Miyamoto

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions. In this study, simultaneous saturation mutagenesis with a restricted set of amino acids at each position was applied to further heighten the activity of the (S)-selective AMDase variant toward α-methyl-α-phenylmalonate. The best variant (V43I/G74C/A125P/V156L/M159L/C188G) showed 9,500-fold greater catalytic efficiency kcat/Km than that of G74C/C188S. Notably, a high level of decarboxylation of α-(4-isobutylphenyl)-α-methylmalonate by the sextuple variant produced optically pure (S)-ibuprofen, an analgesic compound which showed 2.5-fold greater activity than the (R)-selective wild-type AMDase.

Original languageEnglish
Pages (from-to)1965-1971
Number of pages7
JournalBioscience, Biotechnology and Biochemistry
Volume79
Issue number12
DOIs
Publication statusPublished - 2015

Fingerprint

Mutagenesis
Decarboxylation
Enantioselectivity
Ibuprofen
Ports and harbors
Analgesics
Catalytic Domain
Substitution reactions
malonate decarboxylase
Amino Acids

Keywords

  • (S)-profen
  • (S)-selective arylmalonate decarboxylase
  • Activity improvement
  • Saturation mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Applied Microbiology and Biotechnology
  • Analytical Chemistry
  • Organic Chemistry

Cite this

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance. / Yoshida, Shosuke; Enoki, Junichi; Kourist, Robert; Miyamoto, Kenji.

In: Bioscience, Biotechnology and Biochemistry, Vol. 79, No. 12, 2015, p. 1965-1971.

Research output: Contribution to journalArticle

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