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

Looking Back to Amycolatopsis: History of the Antibiotic Discovery and Future Prospects

“…The primary mechanism of resistance to rifampicin (and other rifamycins) consists of rapid selection of resistant mutants (amino acids substitutions) in the rifampicinbinding pocket of RNA polymerase, which results in antibiotic affinity decreasing. Another way to decrease antibiotic affinity is the enzymatic modification of rifamycin by C-21 and C-23 hydroxyl groups [192]. Altogether, these modifications generate the rifamicyn resistome, which negatively affects this class of antibiotics.…”

A Flavin-Dependent Monooxygenase Mediates Divergent Oxidation of Rifamycin

“…Finding RIF SV-O in the enzyme assay of Rif-Orf17 is quite unexpected, because it is the linearized product of RIF SV through C-2 hydroxylation catalyzed by RIF monooxygenase (Rox) in some RIF resistant microorganisms. , The cognate of Rox was also discovered in RIF producers in our recent work . The production of 2 suggests Rif-Orf17 performs phenolic hydroxylation on C-2 to linearize RIF SV in addition to BV oxidation on the 1-carbonyl of RIF S. Although BV oxidation and aromatic hydroxylation are very common reactions catalyzed by FMOs, it is unusual for a single FMO to conduct both oxidations because nucleophilic flavin-4a-peroxide (Fl-O-O – ) and electrophilic flavin-4a-hydroperoxide (Fl-O-OH), respectively, were proposed for them.…”

“…9,10 The cognate of Rox was also discovered in RIF producers in our recent work. 11 The production of 2 suggests Rif-Orf17 performs phenolic hydroxylation on C-2 to linearize RIF SV in addition to BV oxidation on the 1-carbonyl of RIF S. Although BV oxidation and aromatic hydroxylation are very common reactions catalyzed by FMOs, it is unusual for a single FMO to conduct both oxidations because nucleophilic flavin-4aperoxide (Fl-O-O − ) and electrophilic flavin-4a-hydroperoxide (Fl-O-OH), respectively, were proposed for them. Inspecting the fermentation broth of Δorf17 to verify the function of Rif-Orf17, we found linearized product 2 still accumulated (Figure 2IV and Figure S3).…”

“…Inspecting the fermentation broth of Δorf17 to verify the function of Rif-Orf17, we found linearized product 2 still accumulated (Figure 2IV and Figure S3). However, considering a rox analogue was harbored in A. mediterranei outside rif BGC, 11 we postulated that rox and rif-orf17 are both active in the linearization of RIF SV. Indeed, in-frame deletion of rox itself did not block the production of 2 (Figure 2V and Figure S17), while double deletion of rox and rif-orf17 entirely abolished the accumulation of both 1 and 2 (Figure 2VI).…”

“…It was known that RIF S can be easily reduced to RIF SV in the presence of reducing power, and RIF SV can be oxidized to RIF S in the atmosphere. , In the enzyme assays with RIF S as the substrate, RIF SV possibly arose from the the initial substrate through reduction. In accord with this postulation, a significant change in color from reddish brown (RIF S) to yellow (RIF SV) was observed instantly after the addition of the FMNH 2 supply chain.…”