Substrate Flexibility of the Flavin‐Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis. (21st October 2019)

• Record Type: Journal Article
• Authors: Couturier, Maxime; Bhalara, Hiral D.; Chawrai, Suresh R.; Monson, Rita; Williamson, Neil R.; Salmond, George P. C.; Leeper, Finian J.
• Is Part Of: Chembiochem. Volume 21:Number 4(2020)
• Page Start: 523
• Page End: 530
• Publication Date: 2019-10-21
• Subjects: dihydropyrroles -- FAD-dependent oxidase -- mutasynthesis -- prodigiosin -- synthesis; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201900424
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin‐dependent oxidase responsible for the formation of 2‐methyl‐3‐amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5‐methyl‐4‐pentyl‐3, 4‐dihydro‐2 H ‐pyrrole (5 a, resulting from transamination of the aldehyde of 3‐acetyloctanal) and 2‐methyl‐3‐pentyl‐3, 4‐dihydro‐2 H ‐pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a 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 colourimetric assays for PigB and HapB were developed, and the HapB‐catalysed reaction was kinetically characterised. Ten analogues of 5 a 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 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis. Abstract : Seeing red : Prodigiosin has long caught researchers' eyes due to its bright red colour. One of the biosynthetic enzymes in Serratia, PigB, and its homologue in Hahella, HapB, are shown to be FAD‐dependent oxidases that oxidise dihydropyrroles to the corresponding pyrrole. Their substrate specificity has been explored. Dihydropyrroles that are substrates turn Serratia ΔpigD red due to restoration of prodiginine biosynthesis.