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

Abstract

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.

Tetracycline-Inactivating Enzymes

“…Properly defining the distance constraints between flavin-C4a and oxidation sites will enable some predictive capacity. A similar oxidative “soft spot” has been reported for the rifamycin monooxygenase (Rox) that hydroxylates the C2 position of the hydroxynaphthol leading to formation of a 1,2-naphthoquinone ( Koteva et al, 2018 ; Liu et al, 2018 ). In the rifamycin-Rox structure C2 is reported to be 4.7 Å from flavin-C4a.…”

“…Well-known examples of antibiotic destructases include beta-lactamases that hydrolyze the strained 4-membered lactam of beta-lactam antibiotics ( Bush and Jacoby, 2010 ; Brandt et al, 2017 ), and aminoglycoside-inactivating enzymes including phosphotransferases, acetyltransferases, and adenylyltransferases that modify the free amine and hydroxyl groups of aminoglycoside antibiotics ( Ramirez and Tolmasky, 2010 ). Known classes of antibiotic destructases (antibiotic substrates) include peptidases (bogorol, bacitracin) ( Li et al, 2018 ), hydrolases (beta-lactams, macrolides) ( Bush and Jacoby, 2010 ; Morar et al, 2012 ), thioltransferases (fosfomycin) ( Rife et al, 2002 ; Thompson et al, 2013 ), epoxidases (fosfomycin) ( Fillgrove et al, 2003 ), cyclopropanases (colibactin) ( Tripathi et al, 2017 ), acyl transferases (aminoglycosides, chloramphenicol, glufosinate, tabtoxinine-beta-lactam, streptogramin) ( Leslie, 1990 ; Botterman et al, 1991 ; Sugantino and Roderick, 2002 ; Ramirez and Tolmasky, 2010 ; Wencewicz and Walsh, 2012 ; Favrot et al, 2016 ), methyl transferases (holomycin) ( Li et al, 2012 ; Warrier et al, 2016 ), nucleotidylyl transferases (aminoglycosides, lincosamide) ( Morar et al, 2009 ; Ramirez and Tolmasky, 2010 ), ADP-ribosyltransferases (rifamycins) ( Baysarowich et al, 2008 ), glycosyltransferases (aminoglycosides, rifamycins, macrolides) ( Bolam et al, 2007 ; Ramirez and Tolmasky, 2010 ; Spanogiannopoulos et al, 2012 ), phosphotransferases (aminoglycosides, chloramphenicol, rifamycins, macrolides, viomycin) ( Thiara and Cundliffe, 1995 ; Izard and Ellis, 2000 ; Ramirez and Tolmasky, 2010 ; Stogios et al, 2016 ; Fong et al, 2017 ), lyases (streptogramins) ( Korczynska et al, 2007 ), and oxidoreductases (tetracyclines, rifamycins) ( Park et al, 2017 ; Koteva et al, 2018 ). As antibiotic prospecting continues, the list of antibiotic destructases is certain to grow ( Crofts et al, 2017 ; Li et al, 2018 ; Pawlowski et al, 2018 ).…”

The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance

“…Using the available genetic ‘toolkit,’ 7 orthologs of known resistance gene families have been discovered so far in M. abscessus that provide resistance or ‘immunity’ against antibiotics targeting major cellular pathways including cell wall synthesis (β-lactams), RNA synthesis (rifamycins) and protein synthesis (macrolides, aminoglycosides, and tetracyclines). The impressive resistance diversity shown by M. abscessus is expected given its natural habitat is shared by many antibiotic producers, primarily the soil dwelling actinomycetes which are also reservoirs of extensively diverse resistance elements ( Pawlowski et al, 2016 ; Crofts et al, 2017 ; Koteva et al, 2018 ). Exposure to a variety of noxious antimicrobial molecules in the soil might have favored selection of specialized and diverse resistance mechanisms in M. abscessus as well as in other NTM species.…”

Repositioning rifamycins for Mycobacterium abscessus lung disease

“…The M. abscessus genome also contains homologs of flavin adenine dinucleotide (FAD)-dependent monooxygenases [57]. This broad class of enzymes includes the Rox monooxygenases, which inactivate rifampicin in other bacteria [61]. So far, only one M. abscessus FAD-dependent monooxygenase has been characterized: MabTetX (MAB_1496c), a TetX monooxygenase family member, catalyzes the inactivation of tetracycline and doxycycline [17].…”

“…M. abscessus encodes putative FAD-monooxygenases [57], a class of enzymes that confer drug resistance by oxidizing rifamycins. Notably, the Rox monooxygenases of Streptomyces venezuelae and Nocardia farcinica conferred resistance to rifampicin and rifapentine, but not rifabutin [61], as these enzymes can only oxidize rifamycins with a susceptible hydroquinone. It is not known if M. abscessus similarly utilizes enzyme-mediated oxidation to resist rifamycins, but rifabutin would be able to avoid such a resistance mechanism ( Figure 1).…”