4-chlorothreonine and syringomycin

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 α-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 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. s. pv. syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structure-function relationship of halogenases Thr3 and SyrB2 using homology modelling and site-directed mutagenesis.

Topics: Bacterial Proteins; Computational Biology; Molecular Sequence Data; Multigene Family; Mutagenesis, Site-Directed; Pseudomonas syringae; Streptomyces; Structure-Activity Relationship; Threonine

Syringomycin, a lipopeptidolactone assembled from nine amino acid monomers by four enzymes, SyrB1, SyrB2, SyrC, and SyrE, is a cyclic nonribosomal peptide made by plant-associated Pseudomonas spp. This assembly is unusual because the terminal residue, 4-chlorothreonine, has been proposed to be added in trans since the ninth module of the megasynthetase SyrE lacks an adenylation domain required for Thr/Cl-Thr activation. SyrC is now identified as a Thr/Cl-Thr aminoacyltransferase, shuttling the Thr/Cl-Thr moiety between the pantetheinyl arms of the thiolation domain of SyrB1 and the thiolation domain in module nine of SyrE. SyrC uses Cys224 as a catalytic nucleophile to generate a Thr/Cl-Thr-S-enzyme intermediate during transfer. SyrC joins a growing family of such aminoacyl-shuttling enzymes that also use covalent catalysis to move aminoacyl groups from carrier proteins during coumermycin and coronamic acid biosynthesis.

Topics: Aminoacyltransferases; Bacterial Proteins; Biological Transport; Peptide Synthases; Pseudomonas syringae; Threonine