Purification, characterization, and cloning of an S-adenosylmethionine-dependent 3-amino-3-carboxypropyltransferase in nocardicin biosynthesis.
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 beta-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic beta-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.
Reeve
A M
AM
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Breazeale
S D
SD
Townsend
C A
CA
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics / Q000302:isolation & purification*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D007769:Lactams; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D007769:Lactams; C027318:nocardicin; D012436:S-Adenosylmethionine; D019881:Aminoacyltransferases; C115720:S-adenosylmethionine - nocardicin 3-amino-3-carboxypropyltransferase