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 beta-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 k(cat)/K(m) 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 [k(cat)/K(m)=(3.1+/-0.4)x10(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 beta-oxidation of fatty acids.
Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, Groningen, The Netherlands.
Martijn J
MJ
Koetsier
Peter A
PA
Jekel
Marco A
MA
van den Berg
Roel A L
RA
Bovenberg
Dick B
DB
Janssen
D001665:Binding Sites; D020134:Catalytic Domain; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase