Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Cambridge Commonwealth TrustA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate

Substrate flexibility of the flavin-dependent dihydropyrrole oxidases PigB and HapB involved in antibiotic prodigiosin biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5a (resulting from transamination of the aldehyde of 3-acetyloctanal) and 6 (resulting from transamination of the ketone), were synthesised. Only 5a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colorimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5a were synthesised, varying in the C2 and C3 side-chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side-chain tested at C3 were accepted but only short side-chains at C2 were accepted. The knowledge that 5a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5a makes a range of prodigiosin analogues readily available by mutasynthesis.We acknowledge the Frances and Augustus Newman foundation, the Cambridge Commonwealth Trust, Emmanuel College, Cambridge, and the B.B.S.R.C. (award codesBB/N008081/1 and BB/K001833/1) for funding this research