Expression and characterization of (R)-specific enoyl coenzyme A hydratases making a channeling route to...

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4(Pa) from Pseudomonas aeruginosa, termed phaJ4(Pp), was identified. The gene products of phaJ1(Pp), which was identified previously, and phaJ4(Pp) were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4(Pp), as revealed by western blot analysis. These results strongly suggest that PhaJ4(Pp) forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1(Pp) was suggested to be different from that of PhaJ1(Pa) from P. aeruginosa although these two proteins share 67% amino acid sequence identity.