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Recombinant expression and biochemical characterization of Mycobacterium tuberculosis 3Fe-4S ferredoxin Rv1786

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Abstract

Ferredoxins are iron-sulfur protein that mediate electron transfer in cytochrome P450 mono-oxygenase (CYP)-related catalytic reactions in a wide variety of organisms. Rv1786 is a putative ferredoxin, encoded by a gene located downstream of the gene encoding CYP143A1 in the Mycobacterium tuberculosis genome. However, the structure and function of Rv1786 have remained unclear. Here, the recombinant Mtb Rv1786 was expressed, purified as a His-tagged form and characterized with [3Fe-4S] clusters as its cofactors using a series of measurements including SDS-PAGE, western blot, UV/Visible, MALDI-TOF/TOF-MS, and electron paramagnetic resonance spectroscopic analysis. Based on the assessments of surface plasmon resonance (SPR) and steady state kinetic assays, Rv1786 was found to be able to couple with both ferredoxin reductase A (FdrA) and flavoprotein reductase A (FprA) as redox partner, but with a stronger binding to FprA and a better coupling activity to FdrA. Preliminary structural and biochemical characterization of Mtb Rv1786 as a redox partner is presented here.

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Acknowledgments

The authors acknowledge Birgitta Henriques-Normark, Mikael Rhen, Peter Mellroth, and Lu-Ni Chen (Karolinska Institutet) for the critical review, data discussion, and text modification of this manuscript, as well as Yu Lu and Jian Xu for providing the genomic DNA from Mtb H37Rv used in this study.

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  1. Xin-Yi Yang and Xue-Fu You contributed equally to this work

Authors and Affiliations

  1. Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China

    Yun Lu, Feng Qiao, Yue Li, Xiao-Hong Sang, Cong-Ran Li, Jian-Dong Jiang, Xin-Yi Yang & Xue-Fu You

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  1. Yun Lu
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  2. Feng Qiao
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Contributions

XYY and XFY conceived and coordinated the study. YL and XYY designed the major experiments. XFY, CRL, and JDJ provided suggestions on the research plan and contributed ideas to the research proposal. YL carried out the major study work. FQ performed the experiments shown in Tables 3 and 4. YL (Yue Li) contributed to preparation of purified FdrA and FprA. XHS performed the western blot as shown in Fig. 2e. YL, XYY, and XFY analyzed and discussed the data. YL, XYY, and XFY wrote and refined the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Corresponding authors

Correspondence to Xin-Yi Yang or Xue-Fu You.

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Funding

This research was supported by the National Natural Science Foundation of China (NSFC) (81273427 to XYY, 81361138020 to JDJ, and 81573475 to CRL), the National Mega-project for Innovative Drugs (2014ZX09507-009 to XFY), the Beijing Science and Technology Projects (Z141102004414065 and Z151100000315029 to XFY), CAMS Initiative Fund for Innovative Medicine (2016-I2M-3-014 to XFY), and the Peking Union Medical College (PUMC) Youth Fund (33320140177 and 3332016139 to XYY). XYY and CRL were supported by the China Scholarship Council (CSC) State-Sponsored Scholarship Program for Visiting Scholars in Sweden/Europe (CSC2014-3012/201408110091-2).

Conflict of interest

The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Lu, Y., Qiao, F., Li, Y. et al. Recombinant expression and biochemical characterization of Mycobacterium tuberculosis 3Fe-4S ferredoxin Rv1786. Appl Microbiol Biotechnol 101, 7201–7212 (2017). https://doi.org/10.1007/s00253-017-8454-7

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