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 ␤-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic ␤-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 Aagarose, specifically bound the desired transferase to yield the enzyme as a single band of 38 kDa on a silverstained 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.

Two enzymes of diacylglyceryl- O -4′-( N,N,N, -trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides

“…6). Although rare, SAM-dependent 3-amino-3-carboxypropyl transferases are known to participate in other bacterial syntheses, e.g., in the biosynthesis of the antibiotic nocardicine (44).…”

A Novel S-Adenosyl-l-methionine:Arsenic(III) Methyltransferase from Rat Liver Cytosol

“…Aliquots of oxidized supernatants were analyzed by TLC as previously described (30). Fractions that contained the highest levels of the activity that converted arsenite to methylated and dimethylated products were pooled and applied to a column of an S-adenosyl-Lhomocysteine-Sepharose affinity gel that was prepared by the method of Reeve and co-workers (31). The chromatographic gel was fully equilibrated with 50 mM Na phosphate buffer containing 5 mM GSH, 1 mM DTT, and 5% (v/v) glycerol, pH 7.4.…”

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

“…1). The side chain is attached by Nat from AdoMet, to initially present the L-configuration (16). Wilson et al (46) reported a cell-free system from N. uniformis containing both transferase, and epimerase activities.…”

“…Reverse genetics techniques from the AdoMet-dependent 3-amino-3-carboxypropyl transferase (Nat) have allowed the nocardicin biosynthetic gene cluster to be identified and characterized from N. uniformis (16,17). Notably present in the cluster are two non-ribosomal peptide synthetases (NRPSs), genes required for generation of the non-proteinogenic amino acid p-(hydroxyphenyl)glycine, (17) nocL, which encodes a cytochrome P450 responsible for oxime formation in the nocardicins, (18) and nat providing the side chain AdoMet-dependent transferase.…”