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Heterologous Expression and Characterization of a Full-length Protozoan Nitroreductase from Leishmania orientalis isolate PCM2

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Abstract

Leishmaniasis, a parasitic disease found in parts of the tropics and subtropics, is caused by Leishmania protozoa infection. Nitroreductases (NTRs), enzymes involved in nitroaromatic prodrug activation, are attractive targets for leishmaniasis treatment development. In this study, a full-length recombinant NTR from the Leishmania orientalis isolate PCM2 (LoNTR), which causes severe leishmaniasis in Thailand, was successfully expressed in soluble form using chaperone co-expression in Escherichia coli BL21(DE3). The purified histidine-tagged enzyme (His6-LoNTR) had a subunit molecular mass of 36 kDa with no cofactor bound; however, the addition of exogenous flavin (either FMN or FAD) readily increased its enzyme activity. Bioinformatics analysis found that the unique N-terminal sequences of LoNTR is only present in Leishmania where the addition of this region might result in the loss of flavin binding. Either NADH or NADPH can serve as an electron donor to transfer electrons to nitrofurazone; however, NADPH was preferred. Molecular oxygen was identified as an additional electron acceptor resulting in wasteful electrons from NADPH for the main catalysis. Steady-state kinetic experiments revealed a ping-pong mechanism for His6-LoNTR with Km,NADPH, Km,NFZ, and kcat of 28 µM, 68 µM, and 0.84 min−1, respectively. Besides nitroreductase activity, His6-LoNTR also has the ability to reduce quinone derivatives. The properties of full-length His6-LoNTR were different from previously reported protozoa and bacterial NTRs in many respects. This study provides information of NTR catalysis to be developed as a potential future therapeutic target to treat leishmaniasis.

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Acknowledgements

This work was supported by the grant from Kasetsart University Research and Development Institute; KURDI (FF (KU)4.65) (to P. Pimviriyakul), the grant from the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation through grant RGNS 64-030 (to P. Pimviriyakul), the grant from Faculty of Science, Kasetsart University (to P. Pimviriyakul and Y. Kapaothong), and the grant from Development and Promotion of Science and Technology Talents Project (DPST) Scholarship (to T. Tangsupatawat). We thank Assist. Prof. Dr.Teerasak E-Kobon’s laboratory for the gene information.

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  1. Department of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

    Panu Pimviriyakul, Yuvarun Kapaothong & Theerapat Tangsupatawat

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PP: Project administration, Supervision, Conceptualization, Methodology, Investigation, Data analysis, Writing—Original Draft, Review & Editing, Funding acquisition. YK and TT: Methodology, Data curation, Data analysis.

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Pimviriyakul, P., Kapaothong, Y. & Tangsupatawat, T. Heterologous Expression and Characterization of a Full-length Protozoan Nitroreductase from Leishmania orientalis isolate PCM2. Mol Biotechnol 65, 556–569 (2023). https://doi.org/10.1007/s12033-022-00556-3

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