Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis.
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.
Tsuge
Takeharu
T
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8502, Japan. ttsuge@iem.titech.ac.jp
Saito
Yu
Y
Narike
Masaharu
M
Muneta
Koho
K
Normi
Yahaya M
YM
Kikkawa
Yoshihiro
Y
Hiraishi
Tomohiro
T
Doi
Yoshiharu
Y
D000217:Acyltransferases / Q000235:genetics*; D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001704:Biopolymers; D015153:Blotting, Western; D020118:Cupriavidus necator / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011091:Polyesters / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001704:Biopolymers; D017931:DNA Primers; D009841:Oligonucleotides; D011091:Polyesters; D011108:Polymers; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D004262:DNA Restriction Enzymes; D000409:Alanine