title: Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate.
abstract: This study describes protein model of type II Pseudomonas putida GPo1 synthase (PhaC1(Pp)) and using single or multiple points mutagenesis to identify the beneficial amino acid residues that change the PHA accumulation and the substrate chain-length specificity of type II PHA synthase. The P. putida GPp104 PHA⁻ was used as a host for evaluating the substrate specificity and PHA yield of the mutated PhaC1(Pp). The evolved PhaC1(Pp) were coexpressed with β-ketothiolase (phbA(Re)) and the acetoacetyl-CoA reductase (phbB(Re)) to supply sufficient short-chain length (R)-3-hydroxyacyl-CoA as a substrate. A single point mutation at L484V remarkably enhanced the monomer ratio of (R)-3-hydroxybutyrate in a PHA accumulation experiment. Saturation mutagenesis experiment at 484 concluded that Val is the most favorable amino acid in PhaC1(Pp) for incorporating (R)-3-hydroxybutyrate unit synthesis. In addition, a single mutation at Q481M, S482G and A547V obviously increased PHA yields. Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by P. putida GPp104 PHA⁻. This is the first data that spotlighted the important effect of Leu484 on substrate specificity of PHA synthase and Ala547 on the PHA accumulation.
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan; Department of Nursing, Chang Gung University of Science and Technology, Tao-Yuan 33333, Taiwan.
authors.forename: Yi-Jr
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Pei-Chien
authors.initials: PC
authors.lastname: Tsai
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan. Electronic address: clee@ntu.edu.tw.
authors.forename: Chia-Yin
authors.initials: CY
authors.lastname: Lee
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis* / Q000378:metabolism; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D054813:Polyhydroxyalkanoates; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase
keywords: Class II PHA synthase; Polyhydroxyalkanoates; Saturation mutagenesis; Site-directed mutagenesis; Substrate specificity