Rifampin phosphotransferase is an unusual antibiotic resistance kinase

Abstract

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.

Mutational signatures shift induced by chemotherapeutic agents, 5-Fluorouracil and Oxaliplatin, in the gut microbiome

“…Overrepresentation of 3 KEGG pathways involved in metabolism was observed in the 5-FU group with hypergeometric distribution, which involved in the categories "Galactose metabolism" and "Phosphotransferase system (PTS)" (Supplementary Table S6). Enriched genes encoding PTS, which reflected increased representation of multiple carbohydrate transporters, was an important mechanism to confer resistance to selection pressure, such as antibiotic, as reported in the previous studies [21]. In the Oxi group, two KEGG pathways "Ribosome" and "Lysine biosynthesis" were significantly enriched.…”

Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action

“…Resistance to rifamycin antibiotics in the clinic is predominantly due to point mutations in the target, the b subunit of RNA polymerase (Ramaswamy and Musser, 1998), although a rifampin ADP-ribosyltransferase (Baysarowich et al, 2008) (Arr-2) is found in many Gram-negative resistance plasmids. In contrast to the clinic, environmental bacteria have developed a rich diversity of mechanisms to inactivate the rifamycins including ADP-ribosyltransferases (Baysarowich et al, 2008), glycosyltransferases (Spanogiannopoulos et al, 2012), phosphotransferases (Qi et al, 2016;Spanogiannopoulos et al, 2012;Stogios et al, 2016), and monooxygenases (Andersen et al, 1997;Hoshino et al, 2010;Liu et al, 2016). The latter have been detected in a variety of bacteria (Nocardia farcinica, Rhodococcus equi) and are associated with decomposition of the antibiotic.…”

The complex resistomes of Paenibacillaceae reflect diverse antibiotic chemical ecologies

“…Rifampin phosphotransferases (Rph) transfer a phosphate to the C21 position of rifamycins and are widespread in environmental and some pathogenic bacteria, including Listeria monocytogenes [11,33]. This enzyme family is structurally related to phosphoenolpyruvate (PEP) synthases but has a unique domain architecture [33]. Combinations of different rifampin resistance elements in a single strain are known, but not paralogs of Rphs [10,11,34].…”

“…We verified that both predicted rphs (rphC and rphD) confer rifampin resistance when heterologously expressed in E. coli (Table 1). Steady-state kinetic analysis of RphC and RphD revealed that both are catalysts for rifampin phosphorylation, and have a catalytic constant similar to other Rphs [9,11,33] (Supplementary Table 5). Rifampin directly interacts with RpoB by forming four essential hydrogen bonds.…”