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.
Keywords: Enzyme kinetics; Hydration; Iron; Iron trafficking; Nitrile hydratase.