Characterization of a phenylacetate–CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) 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 POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (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 into 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 catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [kcat/Km =(3.1±0.4)×10^2 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 the 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.