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Mutation Effects of a Conserved Alanine (Ala510) in Type I Polyhydroxyalkanoate Synthase from Ralstonia eutropha on Polyester Biosynthesis
Author(s) -
Tsuge Takeharu,
Saito Yu,
Narike Masaharu,
Muneta Koho,
Normi Yahaya M.,
Kikkawa Yoshihiro,
Hiraishi Tomohiro,
Doi Yoshiharu
Publication year - 2004
Publication title -
macromolecular bioscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.924
H-Index - 105
eISSN - 1616-5195
pISSN - 1616-5187
DOI - 10.1002/mabi.200400075
Subject(s) - polyhydroxyalkanoates , ralstonia , saturated mutagenesis , mutant , biochemistry , biosynthesis , polyhydroxybutyrate , chemistry , mutagenesis , escherichia coli , enzyme , stereochemistry , biology , bacteria , gene , genetics
Summary: Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC Re ), have narrow substrate specificity toward ( R )‐3‐hydroxyacyl‐coenzyme A with acyl chain length of C3‐C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC Re was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[( R )‐3‐hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB − 4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB − 4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli . Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild‐type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC Re .Molecular weight distributions of PHA polymers synthesized by the wild‐type enzyme (PhaC Re ) and its mutants.