Biosynthetic pathways for natural products harbor a plethora of unusual enzyme activities. This includes the multi-enzyme assembly lines of modular type I polyketide synthases, which share a common ancestor with the type I fatty acid synthase. In addition to canonical enzyme domains for building-block selection, polyketide extension and reductive modification, some polyketide synthases also include one or more S-adenosylmethionine- (SAM)-dependent methyltransferases. Catalytic activities are most varied and unprecedented among the C-methyltransferases in some polyketide synthase ‘loading modules’, which initiate biosynthesis by assembling a starter unit on an acyl carrier protein (ACP) domain via a thioester linkage to the phosphopantetheine cofactor. The unusual t-butyl group in two natural products – apratoxin A, a Sec61 inhibitor, and bryostatin, a PKC inhibitor – inspired an investigation into its biosynthetic origins. The apratoxin A t-butyl group is formed as pivaloyl-ACP by the loading module of the apratoxin A biosynthetic pathway and by a module within BryX in the bryostatin pathway. These modules contain two methyltransferase domains (MT1 and MT2) of distinctly different sequence and an ACP domain. Pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which provides the initial acyl-transfer step to form malonyl-ACP1. The rare iron-dependent MT1 then catalyzes two methyl transfer reactions to form dimethylmalonyl-ACP2. MT2, which is closely related to the C-methyltransferases of polyketide synthase extension modules3, catalyzes an unusual coupled decarboxylation and third methyl transfer to form pivaloyl-ACP1. The MT1 and MT2 domains flank an inactive, truncated GNAT-like domain (YGNAT, GCN5-related N-AcetylTransferase). In polyketide synthase loading modules with active GNAT-like domains, their sole activity is decarboxylation of malonyl-ACP (curacin A4), methylmalonyl-ACP (saxitoxin), or dimethylmalonyl-ACP (apratoxin, bryostatin, gephyronic acid5). The GNAT-like decarboxylases are selective for their natural substrates. Thus the loading module methyltransferase (1 or 2 methyl transfers) – or lack thereof (0 methyl transfers) – determines the methylation pattern of the module product. A study of the structural basis of 1 methyl transfer in saxitoxin biosynthesis vs. 2 in apratoxin A biosynthesis revealed the unexpected role of an amino acid that allows two methyl transfers if the side chain is Thr, but creates a steric block if the side chain is Ile.
