title: Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance.
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.
authors.affiliation: a Department of Biosciences and Informatics , Keio University , Yokohama , Kanagawa , Japan.
authors.forename: Shosuke
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: a Department of Biosciences and Informatics , Keio University , Yokohama , Kanagawa , Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Enoki
authors.affiliation: b Faculty for Biology and Biotechnology , Ruhr-University Bochum , Bochum , Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Kourist
authors.affiliation: a Department of Biosciences and Informatics , Keio University , Yokohama , Kanagawa , Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Miyamoto
mesh_terms: D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D057927:Hydrophobic and Hydrophilic Interactions; D008314:Malonates / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D008314:Malonates; C030290:malonic acid; D002262:Carboxy-Lyases; C071852:malonate decarboxylase
keywords: (S)-profen; (S)-selective arylmalonate decarboxylase; activity improvement; saturation mutagenesis