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 .
Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacteria tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in 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-F-G). Bioinformatics analysis reveals that SDR family protein Pyr2 functions as a 3-oxoacyl 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 the pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin and enable creating chemical diversity of pyridomycin derivatives.
Pyridomycin is a potent antimycobacterial natural product by specifically inhibiting InhA, a clinically validated antituberculosis drug discovery target. Pyridyl moieties of pyridomycin play an essential role in inhibiting InhA by occupying the reduced form of the nicotinamide adenine dinucleotide (NADH) cofactor binding site. Herein, we biochemically characterize PyrZ that is a multifunctional NadC homologue and catalyzes the successive formation, dephosphorylation, and ribose hydrolysis of nicotinic acid mononucleotide (NAMN) to generate nicotinic acid (NA), a biosynthetic precursor for the pyridyl moiety of pyridomycin. Crystal structures of PyrZ in complex with substrate quinolinic acid (QA) and the final product NA revealed a specific salt bridge formed between K184 and the C3-carboxyl group of QA. This interaction positions QA for accepting the phosphoribosyl group to generate NAMN, retains NAMN within the active site, and mediates its translocation to nucleophile D296 for dephosphorylation. Combining kinetic and thermodynamic analysis with site-directed mutagenesis, the catalytic mechanism of PyrZ dephosphorylation was proposed. Our study discovered an alternative and concise NA biosynthetic pathway involving a unique multifunctional enzyme.
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