title: Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation.
abstract: Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Maria
authors.initials: M
authors.lastname: Cheng
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hayato
authors.initials: H
authors.lastname: Yoshiyasu
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
mesh_terms: D000079:Acetaldehyde / Q000378:metabolism*; D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011767:Pyruvate Decarboxylase / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism
chemical_list: D000095:Acetolactate Synthase; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde; D013835:Thiamine Pyrophosphate