title: Conversion of pyruvate decarboxylase into an enantioselective carboligase with biosynthetic potential.
abstract: Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate into acetaldehyde and CO(2) and requires the cofactors thiamin diphosphate and Mg(2+) for activity. Owing to its catalytic promiscuity and relaxed substrate specificity, PDC catalyzes carboligation side reactions and is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. Although PDC variants with enhanced carboligation efficiency were generated in the past, the native reaction, i.e., formation of aldehydes, is heavily favored over carboligation side reactions in all these biocatalysts. We characterized an active site variant (Glu473Gln) in which partitioning between aldehyde release versus carboligation is inverted with an up to 100-fold preference for the latter pathway. Due to a defective protonation of the central carbanion/enamine intermediate, substrate turnover stalls at this catalytic stage and addition of external aldehydes leads to quantitative and enantioselective formation of 2-hydroxy ketones as shown for (R)-phenylacetyl carbinol, which is afforded with unmatched yields, rates, and purity. This protein variant thus constitutes an example for the rational design of biocatalysts with greatly enhanced accidental catalytic promiscuity by selective blockage of the native reaction and accumulation of reactive intermediates under steady-state turnover conditions.
authors.affiliation: Albrecht-von-Haller-Institute for Plant Sciences and Göttingen Center for Molecular Biosciences, Georg-August-University Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
authors.forename: Danilo
authors.initials: D
authors.lastname: Meyer
authors.forename: Lydia
authors.initials: L
authors.lastname: Walter
authors.forename: Geraldine
authors.initials: G
authors.lastname: Kolter
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D000432:Methanol / Q000138:chemical synthesis; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D005973:Glutamine; D018698:Glutamic Acid; D011767:Pyruvate Decarboxylase; D000432:Methanol