Subha Mukherjee - Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

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  Bougioukou, D. J., Mukherjee, S., and van der Donk, W. A. (2013)   Revisiting the biosynthesis of dehydrophos reveals a   tRNA-dependent pathway, Proc. Natl. Acad. Sci.


    Bioactive natural products containing a C-P bond act as mimics of   phosphate esters and carboxylic acids, thereby competing with   these compounds for active sites of enzymes. Dehydrophos (DHP), a   broad-spectrum antibiotic, is a phosphonotripeptide produced   by   Streptomyces luridus,   in which glycine and leucine are linked to an aminophosphonate   analog of dehydroalanine, ΔAla(P). This unique feature, in   combination with the monomethylation of the phosphonic acid,   renders DHP a Trojan horse type antibiotic because   peptidase-mediated hydrolysis will release methyl   acetylphosphonate, a potent inhibitor of pyruvate dehydrogenase.   Bioinformatic analysis of the biosynthetic gene cluster suggested   that ΔAla(P) would be generated from Ser(P), the phosphonate   analog of Ser, by phosphorylation and subsequent elimination, and   that ΔAla(P) would be condensed with Leu-tRNALeu.   DhpH was anticipated to carry out this elimination/ligation   cascade. DhpH is a multidomain protein, in which a pyridoxal   phosphate binding domain is fused to an   N-acetyltransferase   domain related to the general control nonderepressible-5 (GCN5)   family. In this work, the activity of DhpH was reconstituted in   vitro. The enzyme was able to catalyze the β-elimination reaction   of pSer(P) to generate ΔAla(P), but it was unable to condense   ΔAla(P) with Leu. Instead, ΔAla(P) is hydrolyzed to acetyl   phosphonate, which is converted to Ala(P) by a second pyridoxal   phosphate-dependent enzyme, DhpD. Ala(P) is the substrate for the   condensation with Leu-tRNALeucatalyzed   by the C-terminal domain of DhpH. DhpJ, a   2-oxoglutarate/Fe(II)-dependent enzyme, introduces the vinyl   functionality into Leu-Ala(P) acting as a desaturase, and   addition of Gly by DhpK in a Gly-tRNAGly-dependent   manner completes the in vitro biosynthesis of dehydrophos.

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