title: Engineering the promiscuous racemase activity of an arylmalonate decarboxylase.
abstract: Variant G74C of arylmalonate decarboxylase (AMDase) from Bordatella bronchoseptica has a unique racemising activity towards profens. By protein engineering, variant G74C/V43A with a 20-fold shift towards promiscuous racemisation was obtained, based on a reduced activity in the decarboxylation reaction and a two-fold increase in the racemisation activity. The mutant showed an extended substrate range, with a 30-fold increase in the reaction rate towards ketoprofen. Molecular dynamics simulations and the substrate profile of the racemase indicate that the steric and polar effects of the substrate structure play a more dominant role on catalysis than mere kinetic α-proton acidity. The observation that the conversion of β,γ-unsaturated carboxylic acids does not lead to a rearrangement to form their α,β isomers indicates a concerted rather than a stepwise mechanism. Interestingly, a substrate bearing a nitro group instead of the carboxylic acid group on the α-carbon atom was also converted by the racemase.
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, 2238522 Yokohama, Japan.
authors.forename: Robert
authors.initials: R
authors.lastname: Kourist
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Miyauchi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Uemura
authors.forename: Kenji
authors.initials: K
authors.lastname: Miyamoto
mesh_terms: D016950:Bordetella bronchiseptica / Q000201:enzymology* / Q000235:genetics; D002087:Butyrates / Q000737:chemistry; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003198:Computer Simulation; D014644:Genetic Variation; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D002087:Butyrates; C479121:3-butenoic acid; D002262:Carboxy-Lyases; C071852:malonate decarboxylase; D019998:Racemases and Epimerases