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Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance

Giulia Manina 1, 2, 3 Marco Bellinzoni 2 Maria Rosalia Pasca 1 João Neres 3 Anna Milano 1 Ana Luisa de Jesus Lopes Ribeiro 1, 4 Silvia Buroni 1 Henrieta Škovierová 5 Petronela Dianišková 5 Katarína Mikušová 5 Jozef Marák 5 Vadim Makarov 6 David Giganti 2 Ahmed Haouz 7 Anna Paola Lucarelli 1 Giulia Degiacomi 1 Aurora Piazza 1 Laurent Chiarelli 1 Edda de Rossi 1 Elena Salina 6 Stewart Cole 3 Pedro Alzari 2 Giovanna Riccardi 1, *
* Corresponding author
1 Università degli Studi di Pavia
2 Biochimie Structurale
3 EPFL - Ecole Polytechnique Fédérale de Lausanne
4 UPO - Università degli Studi del Piemonte Orientale - Amedeo Avogadro
5 Comenius University in Bratislava
6 RAS - Russian Academy of Sciences [Moscow]
7 Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6)
Abstract : Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-β-d-ribose 2'-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.
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Submitted on : Wednesday, February 10, 2021 - 6:10:12 PM
Last modification on : Monday, January 10, 2022 - 11:44:05 AM

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Giulia Manina, Marco Bellinzoni, Maria Rosalia Pasca, João Neres, Anna Milano, et al.. Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance. Molecular Microbiology, Wiley, 2010, 77 (5), pp.1172-1185. ⟨10.1111/j.1365-2958.2010.07277.x⟩. ⟨pasteur-03137947⟩

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