Purification, characterization, and cloning of an S-adenosylmethionine- dependent 3-amino-3-carboxypropyltransferase in nocardicin biosynthesis

Abstract

S-Adenosylmethionine:nocardicin 3-amino-3-carboxypropyltransferase catalyzes the biosynthetically rare transfer of the 3-amino-3-carboxypropyl moiety from S-adenosylmethionine to a phenolic site in the β-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic β-lactam antibiotic nocardicin A. Whereas a number of conventional methods were ineffective in purifying the transferase, it was successfully obtained by two complementary affinity chromatography steps that took advantage of the two substrate-two product reaction scheme. S- Adenosylhomocysteine-agarose selected enzymes that utilize S- adenosylmethionine, and a second column, nocardicin A-agarose, specifically bound the desired transferase to yield the enzyme as a single band of 38 kDa on a silver-stained SDS-polyacrylamide gel. The transferase is active as a monomer and exhibits sequential kinetics. Further kinetic characterization of this protein is described and its role in the biosynthesis of nocardicin A discussed. The gene encoding this transferase was cloned from a sublibrary of Nocardia uniformis DNA. Translation gave a protein of deduced mass 32,386 Da which showed weak homology to small molecule methyltransferases. However, three correctly disposed signature motifs characteristic of these enzymes were observed.