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The Fe-type nitrile hydratase from Rhodococcus equi TG328-2 forms an alpha-activator protein complex

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Abstract

Abstract

An Fe-type nitrile hydratase α(ɛ) protein complex from Rhodococcus equi TG328-2 (ReNHase) was discovered and shown by MALDI-TOF to form a 1:1 complex. As isolated, the α(ɛ) protein complex exhibited no detectable NHase activity even in the presence of iron. The addition of the ReNHase β-subunit and Fe(II) to the ReNHase apo-α(ε) complex, provided an enzyme with a kcat value of 0.7 ± 0.1 s−1 using acrylonitrile as the substrate, indicating that the β-subunit is important for the reconstitution of NHase activity. The addition of the reducing agent TCEP enhanced the activity by more than 50% (kcat of 1.7 ± 0.2 s−1). As the (ɛ) protein was previously shown to bind and hydrolyze GTP, the addition of GTP to the as-purified α(ε) complex provided a kcat value of 1.1 ± 0.2 s−1, in the presence of Fe(II) and β-subunit. The addition of TCEP to this combination further enhanced the activity (kcat of 2.1 ± 0.3 s−1). Apo α-subunit was expressed in purified and added to the (ɛ) protein and β-subunits plus Fe(II) and TCEP resulting in a kcat value of 0.7 ± 0.2 s−1 suggesting an α(ɛ) complex can form in vitro. The addition of GTP to this sample increased the observed rate of nitrile hydration by ~ 30%, while TCEP free samples exhibited no activity. Taken together, these data provide insight into the role of the (ɛ) protein and the newly discovered α(ɛ) complex in NHase metallocenter assembly.

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Acknowledgments

We thank the National Science Foundation (CHE-1808711, RCH & BB) for funding this research.

Funding

This work was supported by the National Science Foundation (CHE-1808711 BB & RCH), the Todd Wehr Foundation, and Bruker Biospin.

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Authors and Affiliations

  1. Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA

    K. P. Wasantha Lankathilaka & Richard C. Holz

  2. Department of Physics, Marquette University, 540 N. 15th St, Milwaukee, WI, 53233, USA

    Brian Bennett

  3. Department of Chemistry, Colorado School of Mines, Golden, CO, 80401, USA

    Richard C. Holz

Authors
  1. K. P. Wasantha Lankathilaka
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  2. Brian Bennett
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  3. Richard C. Holz
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Contributions

KPWL prepared expression plasmid, carried out protein expression, purification, enzymatic assays and prepared samples for metal analysis, and analyzed the results. RCH conceived of the idea and wrote the paper with KPWL and BB.

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Correspondence to Richard C. Holz.

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Lankathilaka, K.P.W., Bennett, B. & Holz, R.C. The Fe-type nitrile hydratase from Rhodococcus equi TG328-2 forms an alpha-activator protein complex. J Biol Inorg Chem 25, 903–911 (2020). https://doi.org/10.1007/s00775-020-01806-y

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  • DOI: https://doi.org/10.1007/s00775-020-01806-y

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