Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin

Abstract

Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacterium tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in the pyridomycin core system is an important pharmacophoric group, and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-PyrF-PyrG). Bioinformatics analysis reveals that short-chain dehydrogenase/reductase (SDR) family protein Pyr2 functions as a 3-oxoacyl acyl carrier protein (ACP) reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr-based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin, and enable the creation of chemical diversity of pyridomycin derivatives. IMPORTANCE Tuberculosis (TB) is one of the world's leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacterium tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilizing a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand our understanding of the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.

Keywords: antimycobacterial activity; biosynthesis; enoyl ester; pyridomycin; trans ketoreductase.

FIG 1

Structures of pyridomycin congeners and the pyridomycin biosynthetic gene cluster. (A) Structures of pyridomycin (1), dihydropyridomycins (2 and 3), and pyridomycin B (4). (B) Genetic organization of the pyridomycin biosynthetic gene cluster from S. pyridomyceticus B2517. Pyr2 is highlighted in black.

FIG 2

Bioinformatics analysis of Pyr2. (A) Sequence alignment of selected Pyr2 homologs. The conserved motif in Rossmann fold is highlighted in dashes. The catalytic residues are shown with asterisks. (B) A sequence similarity network (SSN) of Pyr2 homologs (sequence identity of >40%) visualized by Cytoscape. Pyr2 groups are circled with dashes.

FIG 3

In vivo characterization of pyr2 in pyridomycin biosynthesis. (A) UV at 305 nm from HPLC analysis of metabolites from wild-type S. pyridomyceticus B2517, HTT12 (Δpyr2), and HTT12::pyr2. (B) Schematic representation for the gene replacement of pyr2 in S. pyridomyceticus B2517 by insertion of a kanamycin resistance cassette. PCR verification of wild-type pyr2 strain (0.97 bp) and double crossover Δpyr2 mutant (1.70 bp) genotypes. (C) Bioassay against M. smegmatis mc²155 and UV spectrum of the peak from HTT12, pyridomycin as a control. MS/MS spectrum of the peak from HTT12 (D) and pyridomycin (E).

FIG 4

LC-MS analysis of S. pyridomyceticus recombinant strains. Extracted ion chromatograms (EIC, m/z at 409 and 411) from LC-MS analysis of metabolites from S. pyridomyceticus recombinant strains. The detection of each strain was individually scanned by using MS/MS fragmentation patterns of m/z 409 for pyridomycin and 411 for pyridomycin B.

FIG 5

Proposed biosynthesis of pyridomycin supported by the isolation of pyridomycin B from S. pyridomyceticus mutant HTT12.