3-hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea:: Isolation and characterization

In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a K-m = 14.7 mu M for 3-hydroxybenzoic acid, 8.5 mu M for coenzyme A and 229 mu M for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of M-r 41500 and 40500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation, 3HBL had a native M-r of approx, 50000 and is thus active as a monomer.