3-Hydroxy-2-methylbutyrate (3H2MB) has been identified as a minor component of polyhydroxyalkanoates (PHAs) synthesized by bacteria living in activated sludge. In this study, we found that PHA synthase derived from Aeromonas caviae (PhaC Ac ) polymerizes 3H2MB. By expressing PhaC Ac in recombinant Escherichia coli LS5218 and growing cells in the presence of tiglic acid, a PHA copolymer [P(3HB-co-3H2MB)], mainly consisting of 3-hydroxybutyrate (3HB) and up to 37 mol% 3H2MB, was obtained. Analysis of the thermal properties of this novel copolymer indicates that incorporation of 3H2MB into P(3HB) sequence reduced the glass transition temperature (T g ), melting temperature (T m ), and melting enthalpy (DH m ). The cold crystallization temperature (T cc ) was also lowered by incorporating 7 or 23 mol% 3H2MB, in contrast to the findings for other PHA copolymers. This result suggests that P(3HB-co-3H2MB) copolymers are easier to crystallize than P(3HB) and other PHAs. Thus, 3H2MB provides promising new opportunities to generate 3HB-based polymers with novel thermal properties.

d(R)-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid ␤-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1 Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1 Pa from Pseudomonas aeruginosa, despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1 Pp , the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1 Pa , the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1 Pp and PhaJ1 Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1 Pp than in PhaJ1 Pa . Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1 Pp and PhaJ1 Pa , all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1 Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1 Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity. P olyhydroxyalkanoates (PHAs), which are synthesized by some bacterial taxa, act as energy and carbon stores for use during starvation and are desirable for use as bio-based polymer materials (1-3). PHA biosynthesis requires a number of specific enzymes, such as PHA synthases and monomer-supplying enzymes. PHA synthase polymerizes the (R)-3-hydroxyalkanoate moiety of (R)-3-hydroxyacyl coenzyme A (3HA-CoA) into PHA, whereas monomer-supplying enzymes synthesize (R)-3HA-CoA from various metabolic intermediates. To control the material properties of PHAs, it is necessary to regulate the PHA monomer composition, which depends strongly on the enzymatic properties of PHA synthases and monomer-supplying enzymes.(R)-Specific enoyl-CoA hydratase [PhaJ, (R)-hydratase] is a monomer-supplying enzyme that was first found to be involved in the biosynthesis of PHA from fatty acids in Aeromonas caviae (4, 5). Trans-2-enoyl coenzyme A (enoyl-CoA), an intermediate in fatty acid ␤-oxidation, undergoes stereospecific hydration by the function of PhaJ, resulting in the formation of (R)-3HA-CoAs. Currently, four types of phaJ genes (phaJ1 Pa to phaJ4 Pa ) have been identified in the genome of Pseudomonas aeruginosa DSM1707; their translational products were characterized...