Structural Evidence for Rifampicin Monooxygenase Inactivating Rifampicin by Cleaving Its Ansa-Bridge

Abstract

Rifampicin monooxygenase (RIFMO) decreases the potency of rifampicin (RIF) by converting it to oxidative products. Further decomposition of RIF has been observed in bacteria producing RIFMO and contributes to RIFMO-mediated drug resistance. Here we report the first crystal structure of RIFMO in complex with the hydroxylated RIF product. The 2.10 Å resolution structure reveals a breach of the ansa aliphatic chain of RIF between naphthoquinone C2 and amide N1. Our data suggest that RIFMO catalyzes the hydroxylation of RIF at the C2 atom followed by cleavage of the ansa linkage, which leads to inactivation of the antibiotic by preventing key contacts with the RNA polymerase target.

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.…”

“…While the precise degradation products remain unknown for both the enzymatic oxidation and the following non-enzymatic degradation cascade, these mechanistic proposals may serve as useful models as more information becomes available en route to the elucidation of the enzymatic degradation of tetracycline antibiotics ( Yang et al, 2004 ; Forsberg et al, 2015 ; Ghosh et al, 2015 ; Park et al, 2017 ). It is noteworthy that a similar cascade event takes place for the Rox-mediated inactivation of rifamycin where oxidation of the C2 position of the hydroxynaphthalene leads to ring opening of the macrolactam and subsequent linearization of rifamycin ( Koteva et al, 2018 ; Liu et al, 2018 ). A detailed understanding of enzymatic and non-enzymatic degradation cascades for tetracycline and other antibiotics is critical for designing future generations of molecules that overcome these resistance mechanisms and diagnostic tools to detect active antibiotic-inactivating enzymes in clinical samples.…”

“…The tetracycline destructases are FMOs that confer resistance to these next-generation tetracyclines via covalent inactivation ( Moore et al, 2005 ; Grossman et al, 2012 ; Sutcliffe et al, 2013 ; Volkers et al, 2013 ). Antibiotic oxidation is an emerging inactivation resistance strategy that has only been observed for one other antibiotic class, the rifamycins ( Abdelwahab et al, 2016 ; Liu et al, 2016 , 2018 ; Koteva et al, 2018 ). Resistance to rifamycin via enzymatic inactivation is not limited to FMOs; in fact, known rifamycin destructases include FMOs ( Abdelwahab et al, 2016 ; Liu et al, 2016 , 2018 ; Koteva et al, 2018 ), glycosyltransferases ( Spanogiannopoulos et al, 2012 ), ADP-ribosyltransferases ( Baysarowich et al, 2008 ), and phosphotransferases ( Stogios et al, 2016 ).…”

Characterization of the Rifamycin-Degrading Monooxygenase From Rifamycin Producers Implicating Its Involvement in Saliniketal Biosynthesis

“…Meanwhile, our time-course analysis of Rif SV degradation revealed that Rif S was produced during the air oxidation of Rif SV and then consumed in the assays (Supplementary Figure S10c). Previous mechanisms of Rif degradation suggest that a C-1 phenolic hydroxyl group is required to trigger the monooxygenation (Koteva et al, 2018;Liu et al, 2018). Specifically, the deprotonation of the C-1 hydroxyl group is not initiated by a catalytic base from Rox but facilitated by the hydrogen bond network between the Rif C-1/C-8 hydroxyl groups and the N5 atom of the reduced FAD cofactor.…”

Acquisition and Spread of Antimicrobial Resistance: A tet(X) Case Study

“…These broad spectra are also reflected in the ability to destroy a range of antimicrobials, which presently include tetracyclines, rifampicins, sulfonamides, and β-lactams [32]. Besides tetracyclines, which are discussed here, these targets, for example, are represented by aromatic polyketides such as rifampicins [33][34][35]. Another class B FMO can oxidise the carbonyl moiety of the β-lactam ring, thus conferring resistance to imipenem [36].…”