Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum
Résumé
Enzymatic activation of phenylacetic acid (PAA) to phenylacetyl-CoA is an important step in the biosynthesis of the β-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and phenoxyacetic acid (POA) act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a phenylacetate-CoA ligase, was cloned in Escherichia coli as a maltose binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA to phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with kcat/Km values of 0.23 ± 0.06 and 7.8 ± 1.2 mM-1 s-1 for PAA and POA, respectively. Surprisingly, PCL was very efficient in catalyzing the conversion of trans-cinnamic acids to the corresponding CoA thioesters (kcat/Km = (3.1 ± 0.4) • 102 mM-1 s-1 for trans-cinnamic acid). Of all the substrates screened, medium chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty acid activating ancestral enzyme that may have been involved in the β-oxidation of fatty acids.
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