Contribution of the Distal Pocket Residue to the Acyl-Chain-Length Specificity of ( R )-Specific Enoyl-Coenzyme A Hydratases from Pseudomonas spp

Abstract

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...

Within Host Evolution Selects for a Dominant Genotype of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity

“…Two of these variants were nSNPs present in echA6 or eccB5 . The gene echA6 encodes a putative enoyl-CoA hydratase capable of supplying energy and carbon from fatty acid β-oxidation during starvation[31]. EccB5 is a membrane protein of the ESX-5 type VII secretion system involved in secretion of proteins and uptake of nutrients[32].…”

The C‐terminal end of mycobacterial HadBC regulates AcpM interaction during the FAS‐II pathway: a structural perspective

“…The missing residues were added to both the mtb HadBC dimers (considered as monomers) and energy minimized using gromacs . Each monomer and mtb HadAB (https://doi.org/10.2210/pdb4RLJ/pdb) were submitted to the CASTp server with a probe radius of 1.7 Å to calculate the molecular surface for acyl chain binding [54]. The obtained results were viewed using chimerax [55].…”

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene

“…This is because E. coli inherently has a weak ability to supply medium chain-length monomers by modifying β-oxidation products 36) and Pseudomonas-type PhaCs are likely to accumulate PHA under such conditions. 37,38) When co-expressing phaJ YB4 , the PHA accumulation level was increased to 22 wt%, due to PhaJ YB4 's function. As for the comonomer composition, the 3HHx fraction was the highest among 3HA units ranging from C4 to C12, and its relative content reached 63 mol%.…”