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

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 ß-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 ß-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 ß-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 ß-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.

Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy.

Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for "green" chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation.

Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies.

Iron-type nitrile hydratases (NHases) contain an Fe(III) ion coordinated in a characteristic "claw setting" by an axial cysteine thiolate, two equatorial peptide nitrogens, the sulfur atoms of equatorial cysteine-sulfenic and cysteine-sulfinic acids, and an axial water/hydroxyl moiety. The cysteine-sulfenic acid is susceptible to oxidation, and the enzyme is traditionally prepared using butyric acid as an oxidative protectant. The as-prepared enzyme exhibits a complex electron paramagnetic resonance (EPR) spectrum due to multiple low-spin (S = 1/2) Fe(III) species. Four distinct signals can be assigned to the resting active state, the active state bound to butyric acid, an oxidized Fe(III)-bis(sulfinic acid) form, and an oxidized complex with butyric acid. A combination of comparison with earlier work, development of methods to elicit individual signals, and design and application of a novel density functional theory method for reproducing g tensors to unprecedentedly high precision was used to assign the signals. These species account for the previously reported EPR spectra from Fe-NHases, including spectra observed upon addition of substrates. Completely new EPR signals were observed upon addition of inhibitory boronic acids, and the distinctive g1 features of these signals were replicated in the steady state with the slow substrate acetonitrile. This latter signal constitutes the first EPR signal from a catalytic intermediate of NHase and is assigned to a key intermediate in the proposed catalytic cycle. Earlier, apparently contradictory, electron nuclear double resonance reports are reconsidered in the context of this work.

The iron-type nitrile hydratase activator protein is a GTPase.

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a kcat value of 1.2 × 10-3 s-1 and a Km value of 40 µM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 µM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.