ABSTRACT
Phosphonates are rare and unusually bioactive natural products. However, most bacterial phosphonate biosynthetic capacity is dedicated to tailoring cell surfaces with molecules like 2-aminoethylphosphonate (AEP). Although phosphoenolpyruvate mutase (Ppm)-catalyzed installation of C-P bonds is known, subsequent phosphonyl tailoring (Pnt) pathway steps remain enigmatic. Here we identify nucleotidyltransferases in over two-thirds of phosphonate biosynthetic gene clusters, including direct fusions to ~60% of Ppm enzymes. We characterize two putative phosphonyl tailoring cytidylyltransferases (PntCs) that prefer AEP over phosphocholine (P-Cho) - a similar substrate used by the related enzyme LicC, which is a virulence factor in Streptococcus pneumoniae. PntC structural analyses reveal steric discrimination against phosphocholine. These findings highlight nucleotidyl activation as a predominant chemical logic in phosphonate biosynthesis and set the stage for probing diverse phosphonyl tailoring pathways.
Subject(s)
Aminoethylphosphonic Acid/metabolism , Bacteria/metabolism , Bacterial Proteins/metabolism , Biosynthetic Pathways/physiology , N-Acylneuraminate Cytidylyltransferase/metabolism , Organophosphonates/metabolism , Actinobacteria , Bacteria/genetics , Bacterial Proteins/genetics , Cell Wall/metabolism , Crystallization , Crystallography, X-Ray , Escherichia coli , N-Acylneuraminate Cytidylyltransferase/genetics , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Phospholipids/metabolism , Phosphorylcholine/metabolism , Phosphotransferases (Phosphomutases) , Polysaccharides/metabolism , Substrate SpecificityABSTRACT
Rifamycin monooxygenases (Rox) are present in a variety of environmental bacteria and are associated with decomposition of the clinically utilized antibiotic rifampin. Here we report the structure and function of a drug-inducible rox gene from Streptomyces venezuelae, which encodes a class A flavoprotein monooxygenase that inactivates a broad range of rifamycin antibiotics. Our findings describe a mechanism of rifamycin inactivation initiated by monooxygenation of the 2-position of the naphthyl group, which subsequently results in ring opening and linearization of the antibiotic. The result is an antibiotic that no longer adopts the basket-like structure essential for binding to the RNA exit tunnel of the target RpoB, thereby providing the molecular logic of resistance. This unique mechanism of enzymatic inactivation underpins the broad spectrum of rifamycin resistance mediated by Rox enzymes and presents a new antibiotic resistance mechanism not yet seen in microbial antibiotic detoxification.