title: Modification of chimeric (2S, 3S)-butanediol dehydrogenase based on structural information.
abstract: A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 Å. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Shimegi
authors.forename: Kaito
authors.initials: K
authors.lastname: Mochizuki
authors.forename: Takuji
authors.initials: T
authors.lastname: Oyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohtsuki
authors.forename: Masami
authors.initials: M
authors.lastname: Kusunoki
authors.affiliation: Graduate School of Medical and Engineering, Univ. of Yamanashi, Takeda, Kofu 400-8511, Yamanashi, Japan. ui@yamanashi.ac.jp.
authors.forename: Sadaharu
authors.initials: S
authors.lastname: Ui
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007709:Klebsiella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase