C-Methylation of S-adenosyl-L-Methionine Occurs Prior to Cyclopropanation in the Biosynthesis of 1-Amino-2-Methylcyclopropanecarboxylic Acid (Norcoronamic Acid) in a Bacterium

Abstract

Many pharmacologically important peptides are bacterial or fungal in origin and contain nonproteinogenic amino acid (NPA) building blocks. Recently, it was reported that, in bacteria, a cyclopropane-containing NPA 1-aminocyclopropanecarboxylic acid (ACC) is produced from the L-methionine moiety of S-adenosyl-L-methionine (SAM) by non-canonical ACC-forming enzymes. On the other hand, it has been suggested that a monomethylated ACC analogue, 2-methyl-ACC (MeACC), is derived from L-valine. Therefore, we have investigated the MeACC biosynthesis by identifying a gene cluster containing bacterial MeACC synthase genes. In this gene cluster, we identified two genes, orf29 and orf30, which encode a cobalamin (B12)-dependent radical SAM methyltransferase and a bacterial ACC synthase, respectively, and were found to be involved in the MeACC biosynthesis. In vitro analysis using their recombinant enzymes (rOrf29 and rOrf30) further revealed that the ACC structure of MeACC was derived from the L-methionine moiety of SAM, rather than L-valine. In addition, rOrf29 was found to catalyze the C-methylation of the L-methionine moiety of SAM. The resulting methylated derivative of SAM was then converted into MeACC by rOrf30. Thus, we demonstrate that C-methylation of SAM occurs prior to cyclopropanation in the biosynthesis of a bacterial MeACC (norcoronamic acid).

Biosynthesis of Oxetanocin-A Includes a B12-Dependent Radical SAM Enzyme That Can Catalyze both Oxidative Ring Contraction and the Demethylation of SAM

“…Determination of the OxsB crystal structure revealed juxtaposition of the [4Fe-4S]-SAM and the Cbl cofactors in the active site, suggesting that both may participate directly in the catalytic cycle of OxsB . Thus far, a total of 12 B 12 -dependent radical SAM enzymes (TsrM, − Fom3, − PhpK, − ThnK, TokK, CysS, , PoyC, GenK, , GenD1, MaMmp10, orf29 in bacterial MeACC synthase, and OxsB 3 ) have been studied in vitro. Unlike other members of this group, which are all involved in the methylation of unactivated carbons or phosphinate centers, OxsB is the only enzyme in this class known to catalyze an oxidative ring contraction .…”

Overall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis

“…These enzymes contain both a cobalamin (B 12 )-binding domain and a domain characteristic of the radical SAM superfamily, which uses a [4Fe-4S] cluster and S -adenosyl- l -methionine (SAM) to initiate a diverse range of different chemical transformations . Class B rSAM methyltransferases attach methyl groups to unactivated carbon centers during the biosynthesis of a variety of molecules, such as gentamicin, thienamycin, cystobactamids, polytheonamides, , norcoronamic acid, and methyl-coenzyme M reductase. , Although in vitro activity has been obtained for several of these enzymes, many others remain uncharacterized and many more predicted sequences have yet to be assigned a function. , Investigating the Fom3 reaction may therefore provide insights into many other important biochemical transformations.…”

Biosynthesis of cyclopropane in natural products

“…A subsequent in vivo gene deletion study and in vitro enzyme characterization suggests a revised biosynthetic path for norCMA production from L-met (rather than from L-Val). 46 As shown in Fig. 7B, Orf30 was found to catalyze the C-methylation reaction of Met, and this is followed by the Orf29-catalyzed cyclopropanation in a way which is analogous to that of GnmY.…”

“…45 A recent heterologous expression study on a novel NRPS gene cluster sheltering the homologs of Swb7 and Swb6 (denoted as Orf29 and Orf30, respectively) led to the production of Q6402A. 46 The Orf29 (as well as Swb7) belong to cobalamin (B 12 )-dependent radical SAM methyltransferase family, whereas Orf30 (as well as Swb6), show sequence homology similar to that of the ACC synthase GnmY. A subsequent in vivo gene deletion study and in vitro enzyme characterization suggests a revised biosynthetic path for norCMA production from L-met (rather than from L-Val).…”