Monooxygenation of rifampicin catalyzed by the rox gene product of Nocardia farcinica: structure elucidation, gene identification and role in drug resistance

Hoshino, Yasutaka; Fujii, Shoko; Shinonaga, Hideki; Arai, Kunijiro; Saito, Fumiko; Fukai, Toshio; Satoh, Hiroyuki; Miyazaki, Yoshitsugu; Ishikawa, Jun

doi:10.1038/ja.2009.116

Cited by 43 publications

(59 citation statements)

References 13 publications

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“…These features are indicative of a well folded flavoenzyme. These results confirm that RIFMO is a flavoenzyme, which is consistent with the prediction based on sequence analysis (13).…”

Section: Resultssupporting

confidence: 80%

“…The enzyme was discovered from pathogenic Nocardia farcinica (15). Phylogenetic analysis suggests that RIFMO is distinguishable from other known flavoprotein monooxygenases (13). A highly similar gene (iri) that confers moderate RIF resistance was also found in Rhodococcus equi (12).…”

mentioning

confidence: 98%

“…RIFMO decreases the potency of RIF by converting it to 2Ј-N-hydroxy-4-oxo-rifampicin (RIF-O) ( Fig. 1), which has a markedly higher minimal inhibition concentration against a spectrum of Gram-negative and Gram-positive bacteria (13). The addition of a hydroxyl group on the N2Ј atom of the piperazine moiety, which is absent in other rifamycins, leads to decolorization of RIF (i.e.…”

mentioning

confidence: 99%

“…At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12,13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl.…”

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confidence: 99%

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The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

Liu¹,

Abdelwahab

Campo

et al. 2016

Journal of Biological Chemistry

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Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 M. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. Rifampicin (RIF)3 ( Fig. 1) is a potent frontline antibiotic against tuberculosis and other mycobacterial infections, but extensive usage of RIF and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity (1, 2). In addition to the point mutations in RNA polymerase that are responsible for resistance in mycobacteria (3, 4), some bacterial species, such as soil actinomycetes and parasitic bacteria, employ secondary enzyme-mediated inactivation mechanisms that chemically modify RIF to less active forms or degradation products (5).At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl. These hydroxyls are important for antibiotic action because they hydrogen-bond to a conserved region of the ␤-subunit in RNA polymerase, which is the target of RIF (14). Covalent modification of RIF hydroxyls with ADP-ribose or phosphate results in high level resistance in Escherichia coli, such as a 64-fold increase in the RIF minimal inhibition concentration (6, 10). Modificat...

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“…These features are indicative of a well folded flavoenzyme. These results confirm that RIFMO is a flavoenzyme, which is consistent with the prediction based on sequence analysis (13).…”

Section: Resultssupporting

confidence: 80%

mentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

Liu¹,

Abdelwahab

Campo

et al. 2016

Journal of Biological Chemistry

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show abstract

“…These covalent modifications occur on the critical hydroxyls of the 1-amino, 2-naphthol, 4-sulfonic acid (ansa) chain of rifamycins and thus make rifamycins unable to fit into the binding pocket on RNAP. Additional resistance mechanisms have been reported also (14)(15)(16).…”

mentioning

confidence: 99%

Structural basis of rifampin inactivation by rifampin phosphotransferase

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.antibiotic resistance | rifampin | phosphotransferase | molecular mechanism | toggle switch

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Genetic Manipulation ofNocardiaSpecies

et al. 2016

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Nocardia spp. are aerobic, Gram-positive, catalase positive, and non-motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities. It can be expected that knowledge of the biochemistry behind their pathogenicity will be crucial in developing effective treatment strategies. These genetic tools can be utilized to develop rational metabolic engineering approaches for crafting host strains with higher production or biotransformation ability.

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Monooxygenation of rifampicin catalyzed by the rox gene product of Nocardia farcinica: structure elucidation, gene identification and role in drug resistance

Cited by 43 publications

References 13 publications

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

Structural basis of rifampin inactivation by rifampin phosphotransferase

Genetic Manipulation ofNocardiaSpecies

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