The Fe-type nitrile hydratase from Rhodococcus equi TG328-2 forms an alpha-activator protein complex
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ORIGINAL PAPER
The Fe‑type nitrile hydratase from Rhodococcus equi TG328‑2 forms an alpha‑activator protein complex K. P. Wasantha Lankathilaka1 · Brian Bennett2 · Richard C. Holz1,3 Received: 9 January 2020 / Accepted: 6 July 2020 © Society for Biological Inorganic Chemistry (SBIC) 2020
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.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00775-020-01806-y) contains supplementary material, which is available to authorized users. * Richard C. Holz [email protected] 1
Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201‑1881, USA
2
Department of Physics, Marquette University, 540 N. 15th St, Milwaukee, WI 53233, USA
3
Department of Chemistry, Colorado School of Mines, Golden, CO 80401, USA
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Vol.:(0123456789)
JBIC Journal of Biological Inorganic Chemistry
Graphic abstract
Keywords Nitrile hydratase · Iron · Hydration · Iron trafficking · Enzyme kinetics
Introduction Nitrile hydratases (NHases, EC 4.2.1.84) are metalloenzymes that catalyze the hydration of nitriles to their corresponding higher value amides under mild conditions (room temperature and physiological pH) [1, 2]. X-ray crystallographic studies indicate that they are α2β2 heterotetramers with an active site consisting of three cysteine residues, two amide nitrogens, a water molecule, and either a low-spin, non-heme Fe(III) ion (Fe-type) or a low-spin, non-corrin Co(III) ion (Co-type) [3, 4]. Two of the active site cysteine residues are post-translationally modified to cysteinesulfinic acid (–SO2H) and cysteine-sulfenic acid (–SOH) yielding an unusual metal coordinat
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