Studies On The NRPS Module PyrG In Pyridomycin Biosynthesis

Pyridomycin is a structurally unique antimycobacterial antibiotic produced by Streptomycespyridomyceticus NRRL B-2517. It is an unusual12-membered cyclo-depsipeptide composed of fourmoieties in the following order: N-3-hydroxypicolinyl-L-threonine,3-(3-pyridyl)-L-alanine,propionic acid, and2-hydroxy-3-methylpent-2-enoic acid which is probably epimerized fromα-keto-β-methylvaleric acid. To the best of our knowledgement, it is the only depsipeptide thatcontains an enolic acid, indcating that a novel biosynthetic pathway will be involved in pyridomycinbiosynthesis.The biosynthetic gene cluster for pyridomycin has been previously cloned and identified. Thebiosynthetic pathway of pyridomycin has been proposed by a series of genetic and biochemicalstudies. PyrG is the last NRPS module in biosynthetic gene cluster of pyridomycin. Special featuresof the PyrG architecture are the location of a PKS KR domain and two A domains.The main goal of this study was to determine the activity of the tandem A domains and thefunction of KR domain in PyrG. Firstly, based on the bioinformatics analysis, we predict that thetandem A domain may act together to activate the substrate because PyrG-A1lack the conserved A3motif for adenylate formation while the A6and A8motifs in PyrG-A2are not conserved. Fourexpression constructs pET28a-PyrG-A1, pET28a-PyrG-A2, pET28a-PyrG-AA (A1A2),pET28a-PyrG-AT (A1A2KRT) were generated and expressed to get soluble proteins for testingenzyme activity by PPi-exchange assay. We found that these two A domains were not acting togetherto activate the substrate and only PyrG-A2and PyrG-AT were functional. In addition, the A domainsof PyrG can accept a range of substrates including leucine, isoleucine, α-keto-β-methylvaleric acid,and α-hydroxy-β-methylvaleric acid derived from isoleucine.The KR domain in PyrG was predicted to be functional through the bioinformatics analysis andit was demonstrated to be essential for pyridomycin production by point mutation and feeding experiment[1]. The in vitro experiments demonstrated that PyrG-KR reduced the α-keto group ofα-keto-β-methylvaleric acid to form hydroxyl group after being tethered to the PCP domain in PyrGmodule, ready for ester bond formation catalyzed by the C domain by using Q-TOF-LC-MStechnique. The PyrH, an MbtH-like protein was used the in vitro experiments to assist the function ofthe A domain in the NRPS module PyrG.This study provides useful information for the elucidation of synthetic mechanism ofpyridomycin and enriches the examples of hybrid PKS and NRPS system. Given the uniquemolecular architecture and potent antimycobacterial activity of pyridomycin, the study ofpyridomycin biosynthesis also offers an opportunity to generate pyridomycin derivatives bycombinatorial biosynthesis.