Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli.
An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E. coli led to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5alpha. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) .
Reiser
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Monsanto Company, Ag Sector, St. Louis, MO 63198, USA.
Mitsky
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Gruys
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KJ
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004746:Enoyl-CoA Hydratase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006880:Hydroxy Acids / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D019301:Oleic Acid; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D020586:Thiocapsa / Q000201:enzymology / Q000235:genetics
D000214:Acyl Coenzyme A; D006880:Hydroxy Acids; D011994:Recombinant Proteins; D019301:Oleic Acid; C010701:crotonyl-coenzyme A; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C406162:R-specific trans-2,3-enoylacyl-CoA hydratase; D004746:Enoyl-CoA Hydratase