Biosynthesis of chloramphenicol in Streptomyces sp. 3022a. Properties of an aminotransferase accepting p-aminophenylalanine as a substrate.

In the presence of alpha-ketoglutarate, cell-free extracts of Streptomyces species 3022a catalysed transfer of the amino group from p-aminophenylalanine, yielding an unstable product similar to that obtained by the action of D- and L-amino acid oxidases on the amino acid. The enzyme, purified 16-fold from cell homogenates by chromatography on ion-exchange celluloses, hydroxyapatite, and cross-linked dextran gel, has a molecular weight of 90 000 and a broad pH optimum at 8.0. It is active with L-phenylalanine and L-tyrosine as well as p-amino-DL-phenylalanine as amino donors, and its relative activity towards these substrates did not change during purification. Polyacrylamide disc-gel electrophoresis of the partially purified enzyme gave single zones with identical mobility when the gels were assayed for activity with p-aminophenylalanine and tyrosine as amino donors. The results indicate that synthesis of p-aminophenylalanine en route to chloramphenicol uses a multispecific aminotransferase for aromatic amino acids. With L-phenylalanine as substrate, the preferred amino-accepting co-substrate was alpha-ketoglutarate. Some kinetic constants for the enzyme were determined, and its requirement for pyridoxal phosphate was demonstrated.