Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP:CoA transacylase, which diverts intermediates of fatty acid de novo biosynthesis
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The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in variousPseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis. PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His6) fusion protein in high yield. The His6-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established. Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His6-PhaG, gaveK0.5 values of 28 μM (ACP) and 65 μM (3-hydroxyacyl-CoA) consideringV max values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz). In addition, the alignment with the α/β-hydrolase fold region indicated that PhaG belongs to α/β-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% α−helix, 22% β-sheet, 18% β-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme. Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.
Journal of Biological Chemistry
This research was originally published in Journal of Biological Chemistry (JBC). Hoffmann et al, Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis, Journal of Biological Chemistry (JBC) Vol. 277, No. 45, pp. 42926–42936, 2002. Copyright the American Society for Biochemistry and Molecular Biology. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive version.
Medicinal and Biomolecular Chemistry not elsewhere classified