Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum.
The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.
Alvarez
E
E
Departamento de Microbiología, Facultad de Biología, Universidad de León, Spain.
Cantoral
J M
JM
Barredo
J L
JL
Díez
B
B
Martín
J F
JF
D000217:Acyltransferases / Q000302:isolation & purification*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010397:Penicillanic Acid / Q000378:metabolism* / Q000031:analogs & derivatives; D010400:Penicillin G / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010407:Penicillium / Q000201:enzymology*; D010408:Penicillium chrysogenum / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D046915:Penicillin-Binding Proteins; D010397:Penicillanic Acid; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; D010400:Penicillin G; C004408:aminopenicillanic acid; D004229:Dithiothreitol