Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation.
The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid ss-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa), to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short- (C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4(Pa) were performed using purified samples. Kinetic analysis revealed that only PhaJ4(Pa) exhibits almost constant maximum reaction rates (V(max)) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.
Tsuge
Takeharu
T
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. ttsuge@iem.titech.ac.jp
Taguchi
Kazunori
K
Seiichi
Taguchi
T
Doi
Yoshiharu
Y
D000473:Alkanes / Q000737:chemistry*; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013997:Time Factors
D000473:Alkanes; D005227:Fatty Acids; D004746:Enoyl-CoA Hydratase; D010100:Oxygen