title: Characterization of site-specific mutations in a short-chain-length/medium-chain-length polyhydroxyalkanoate synthase: in vivo and in vitro studies of enzymatic activity and substrate specificity.
abstract: Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases.
authors.affiliation: Enzyme Research Team, Biomass Engineering Program, RIKEN, Saitama, Japan.
authors.forename: Jo-Ann
authors.initials: JA
authors.lastname: Chuah
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tomizawa
authors.forename: Miwa
authors.initials: M
authors.lastname: Yamada
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Keiji
authors.initials: K
authors.lastname: Numata
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002861:Chromobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D054813:Polyhydroxyalkanoates; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase