title: A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.
abstract: Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.
authors.affiliation: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan, manami.hyakutake@riken.jp.
authors.forename: Manami
authors.initials: M
authors.lastname: Hyakutake
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tomizawa
authors.forename: Kouhei
authors.initials: K
authors.lastname: Mizuno
authors.forename: Tamao
authors.initials: T
authors.lastname: Hisano
authors.forename: Hideki
authors.initials: H
authors.lastname: Abe
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001409:Bacillus cereus / Q000201:enzymology*; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D058105:Polymerization
chemical_list: D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase