caa a22 4500 605506507 CHVBK 20210128100627.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-014-6276-4 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6276-4 A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions [Elektronische Daten] [Manami Hyakutake, Satoshi Tomizawa, Kouhei Mizuno, Tamao Hisano, Hideki Abe, Takeharu Tsuge] 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 (Cys151, Asp306, and His335) 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. Springer-Verlag Berlin Heidelberg, 2014 Bacillus nationallicence Escherichia coli nationallicence Polyhydroxyalkanoate nationallicence Class IV PHA synthase nationallicence Alcoholysis nationallicence Catalytic residue nationallicence Hyakutake Manami Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut Tomizawa Satoshi Department of Biological and Chemical Systems Engineering, Kumamoto National College of Technology, 2627, Hirayamashin-machi, 866-8501, Yatsushiro, Kumamoto, Japan aut Mizuno Kouhei Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, 802-0985, Kokuraminami-ku, Kitakyushu, Japan aut Hisano Tamao RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, 679-5148, Sayo-gun, Hyogo, Japan aut Abe Hideki Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut Tsuge Takeharu Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4701-4711 0175-7598 99:11<4701 2015 99 253 https://doi.org/10.1007/s00253-014-6276-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6276-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hyakutake Manami Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut NATIONALLICENCE 700 1- Tomizawa Satoshi Department of Biological and Chemical Systems Engineering, Kumamoto National College of Technology, 2627, Hirayamashin-machi, 866-8501, Yatsushiro, Kumamoto, Japan aut NATIONALLICENCE 700 1- Mizuno Kouhei Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, 802-0985, Kokuraminami-ku, Kitakyushu, Japan aut NATIONALLICENCE 700 1- Hisano Tamao RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, 679-5148, Sayo-gun, Hyogo, Japan aut NATIONALLICENCE 700 1- Abe Hideki Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut NATIONALLICENCE 700 1- Tsuge Takeharu Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, 226-8502, Midori-ku, Yokohama, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4701-4711 0175-7598 99:11<4701 2015 99 253