A fatty acyl coenzyme A synthetase (FadD) from
Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher
kcat and
Km values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the
fadD gene from
P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3Δ
fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3Δ
fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3Δ
fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of
fadD in
P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid:CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg
2+-dependent enzymes that activate alkanoic acids to CoA esters for β oxidation (Fig. ) (
2,
17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (
34). FadD is a cytoplasmic membrane-associated FACS (
7), with sizes ranging from 47 kDa to 62 kDa (
2,
14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.
Open in a separate windowFadD activation of fatty acids to their CoA derivatives proceeds through ATP-dependent covalent binding of AMP to fatty acid with the release of inorganic pyrophosphate, followed by C-S bond formation to obtain fatty acyl-CoA ester and subsequent release of AMP. FadD requires the presence of Mg
2+ ions to be active (
2,
17).It is known that bacteria such as
Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (
5,
6,
42,
45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in
P. putida U with a high homology to long-chain
fadD products from
Escherichia coli and
Pseudomonas fragi (
6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (
6). In a follow-up study, Olivera et al. showed that the
fadD mutants reverted to wild-type characteristics within 3 days of incubation, indicating that
fadD could be replaced by the activity of a second enzyme (
25). Indeed, two
fadD gene homologues have been identified in
P. putida U, namely,
fadD1 and
fadD2, with
fadD2 being expressed only when
fadD1 is inactivated (
25). A putative FadD in
P. putida KT2440 is encoded by PP_4549 (
24), but the protein has not been studied nor has the effect of
fadD (PP_4549) expression/disruption been examined. In the current study the knockout and complementation of
fadD from
P. putida CA-3 demonstrated that its activity is critical for growth and PHA accumulation with long-chain aromatic and aliphatic alkanoic acids and that the activity is not replaced by a second enzyme. While reports have shown that PHA polymerase greatly affects PHA monomer composition (
30,
40), no evidence of the specific effect of FACS on PHA accumulation so far exists.We describe here the purification, kinetic characterization, gene deletion, and homologous expression of FadD from
P. putida CA-3. This is a fundamental study of the activity and physiological role of FACS activity in aromatic and aliphatic alkanoic acid activation and PHA accumulation.
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