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 language | English |
|---|---|
| Pages (from-to) | 1965-1971 |
| Number of pages | 7 |
| Journal | Bioscience, Biotechnology and Biochemistry |
| Volume | 79 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - 2015 |
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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 journal › Article
}
TY - JOUR
T1 - Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance
AU - Yoshida, Shosuke
AU - Enoki, Junichi
AU - Kourist, Robert
AU - Miyamoto, Kenji
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - (S)-profen
KW - (S)-selective arylmalonate decarboxylase
KW - Activity improvement
KW - Saturation mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=84946943993&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84946943993&partnerID=8YFLogxK
U2 - 10.1080/09168451.2015.1060844
DO - 10.1080/09168451.2015.1060844
M3 - Article
C2 - 26115233
AN - SCOPUS:84946943993
VL - 79
SP - 1965
EP - 1971
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 12
ER -