Insight into the structure-function relationship of the nonheme iron halogenases involved in the biosynthesis of 4-chlorothreonine-Thr3 from Streptomyces sp OH-5093 and SyrB2 from Pseudomonas syringae pv. syringae B301DR

Molecular cloning of the biosynthetic gene cluster involved in the production of free 4-chlorothreonine in Streptomyces sp. OH-5093 showed the presence of six ORFs: thr1, thr2, thr3, orf1, orf2 and thr4. According to bioinformatic analysis, thr1, thr2, thr3 and thr4 encode a free-standing adenylation domain, a carrier protein, an Fe(II) nonheme a-ketoglutarate-dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4-chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from Pseudomonas similar to syringae pv. syringae B301DR. Orf1 and orf2 show sequence similarity with alanyl/threonyl-tRNA synthetases editing domains and drug metabolite transporters, respectively. We show that thr3 can replace the halogenase gene syrB2 in the biosynthesis of syringomycin, by functional complementation of the mutant P.similar to s.similar to pv.similar to syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structurefunction relationship of halogenases Thr3 and SyrB2 using homology modelling and site-directed mutagenesis