Deciphering The Mechanism Of The Pyridomycin Biosynthesis

Pyridomycin is a structurally unique antimycobacterial antibiotic produced by Streptomyces pyridomyceticus NRRL B-2517. It is an unusual 12-membered ring depsipeptide composed of four moieties in the following order: N-3-hydroxypicolinyl-L-threonine, 3-(3-pyridyl)-L-alanine, propionic acid, and 2-hydroxy-3-methylpent-2-enoic acid which is probably epimerized from -keto-?-methylvaleric acid.To facilitate molecular genetic studies of S. pyridomyceticus, the conditions for the conjugal transfer of DNA from E. coli were established and optimized.The biosynthetic gene cluster for pyridomycin has been cloned and identified from pyridomycin producer. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase (PKS) gene.PyrA, a predicted 3-HPA:AMP ligase, which was biochemically characterized to react with ATP in the presence of 3-HPA to release PPi and proved to be involved in the biosynthesis of streptogramin B antibiotics such as pristinamycin I. Gene replacement and complementation definitely demonstrated that PyrA was required for the pyridomycin biosynthesis. Therefore, PyrA can be envisioned to activate 3-HPA mediated by ATP. The in vitro PPi exchange assay demonstrated the flexibility of PyrA. PyrU features the conserved LGxxS motif for phosphopantetheinylation, and the in vivo and in vitro analysis provide clear evidence that PyrU functions as a PCP for loading 3-HPA. We showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein (PCP), PyrU, which functions as a loading module that initiates pyridomycin biosynthesis.A special feature is the presence of a PKS-type ketoreductase (KR) domain embedded in an NRPS. The KR point mutant HTT19PSY (S163A, Y176F) did not produce any pyridomycin intermediate, indicated that the KR domain is functional and essential for pyridomycin production. The tandem A domains of PyrG may act together to activate the substrate and tether it to PCP because PyrG-A1 lacks the conserved A3 motif for adenylate formation and the A6 and A8 motifs in PyrG-A2 are not conserved. -keto-?-methylvaleric acid, derived from isoleucine, is activated by the tandem A domains of NRPS4 and tethered to PCP. The KR domain embedded in PyrG was predicted to be functional because it contains a Rossmann fold for NAD(P)H binding, and conserved Lys, Ser, and Tyr residues. Mutation of conserved active sites of the KR domain (S163A, Y176F) resulted in complete loss of pyridomycin production, indicating that it is necessary for pyridomycin production.Five genes (pyrP-T) are transcribed in the same orientation and may constitute an operon. They initially seemed to be involved in the biosynthesis of pyridyl moieties. However, the inactivation of all these genes did not affect pyridomycin production. These findings indicated that the biosynthesis of the pyridyl moieties would use some genes from the primary metabolism.PyrA could also activate other aromatic acids generating three pyridomycin analogues in vivo by feeding the precusors into the pyridomycin producing strain. These findings provide opportunities to generate pyridomycin derivatives with novel or enhanced bioactivities by rational engineering of the biosynthetic pathway or combinatorial biosynthesis. Given its unique molecular architecture and potent antimycobacterial activity, pyridomycin also offers an opportunity to discover new chemistry for natural product biosynthesis.