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

Abstract

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.

Fig. 1. General structure of the bipyrrole core of the tambjamines (1, R represents acyl chains ranging from C2 to C12), structure of tambjamine YP1 (2) and structure of the proposed amine intermediate that forms the tambjamine YP1 tail (3).

Fig. 2. Proposed pathway for the formation of the tambjamine YP1 C12 amine tail in P. tunicata. This begins with acyl-adenylate formation catalyzed by the N-terminal TamA adenylation domain (ANL, blue) and transfer of the fatty acid to the acyl carrier protein (ACP) domain (purple) which has been converted to the phosphopantetheine (4′-PP) form by a 4′-PP transferase (PPT) using coenzyme A (CoASH). The TamA-bound acyl-thioester is then dehydrogenated by TamT (dehydrogenase, DH) and thioester reduction and transamination is catalyzed by the bifunctional TamH (thioester reductase, TR, transaminase, TA) which releases the long chain amine (the order of the TamT and TamH reactions is unknown).

Fig. 3. Denaturing ESI-MS over the charge states 60+ to 58+ of (a) apo-TamA and (b) holo-TamA showing a mass difference of 513 Da. The native mass spectrum (c) of TamA as-purified shows two species for each charge state (18+ to 16+). The higher abundance species is consistent with a TamA:acyl-adenylate bound complex.

Fig. 4. Denaturing ESI-MS analysis of the 7+ charge state of (a) holo-TamA ACP domain and (b) holo-TamA ACP after incubation with apo-TamA.

Fig. 5. Denaturing ESI-MS analysis of the 7+ charge state of the holo-TamA ACP domain after incubation with holo-TamA, Mg²⁺, ATP and fatty acids ranging in length from C2–C16. The values of the deconvoluted masses are described in Table S2.