Coupled Natural Fusion Enzymes in a Novel Biocatalytic Cascade Convert Fatty Acids to Amines.

Tambjamine YP1 is a pyrrole-containing natural product. Analysis of the enzymes encoded in the Pseudoalteromonas tunicata "tam" biosynthetic gene cluster (BGC) identified a unique di-domain biocatalyst (PtTamH). Sequence and bioinformatic analysis predicts that PtTamH comprises an N-terminal, pyridoxal 5'-phosphate (PLP)-dependent transaminase (TA) domain fused to a NADH-dependent C-terminal thioester reductase (TR) domain. Spectroscopic and chemical analysis revealed that the TA domain binds PLP, utilizes l-Glu as an amine donor, accepts a range of fatty aldehydes (C7-C14 with a preference for C12), and produces the corresponding amines. The previously characterized PtTamA from the "tam" BGC is an ATP-dependent, di-domain enzyme comprising a class I adenylation domain fused to an acyl carrier protein (ACP). Since recombinant PtTamA catalyzes the activation and thioesterification of C12 acid to the holo-ACP domain, we hypothesized that C12 ACP is the natural substrate for PtTamH. PtTamA and PtTamH were successfully coupled together in a biocatalytic cascade that converts fatty acids (FAs) to amines in one pot. Moreover, a structural model of PtTamH provides insights into how the TA and TR domains are organized. This work not only characterizes the formation of the tambjamine YP1 tail but also suggests that PtTamA and PtTamH could be useful biocatalysts for FA to amine functional group conversion.

Synthesis of N-acyl amide natural products using a versatile adenylating biocatalyst.

Natural products are secondary metabolites produced by many different organisms such as bacteria, fungi and plants. These biologically active molecules have been widely exploited for clinical application. Here we investigate TamA, a key enzyme from the biosynthetic pathway of tambjamine YP1, an acylated bipyrrole that is produced by the marine microorganism Pseudoalteromonas tunicata. TamA is a didomain enzyme composed of a catalytic adenylation (ANL) and an acyl carrier protein (ACP) domain that together control the fatty acid chain length of the YP1. Here we show that the TamA ANL domain alone can be used to generate a range of acyl adenylates that can be captured by a number of amines thus leading to the production of a series of fatty N-acyl amides. We exploit this biocatalytic promiscuity to produce the recently discovered class of N-acyl histidine amide natural products from Legionella pneumophila.

The carbon chain-selective adenylation enzyme TamA: the missing link between fatty acid and pyrrole natural product biosynthesis.

The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4'-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.