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.

Abstract: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 NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.Biochemical Journal, Vol 474, No. 2 (January 15, 2017): pg. 247-258. DOI. This article is © Portland Press Limited and permission has been granted for this version to appear in e-Publications@Marquette. Portland Press Limited does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Portland Press Limited. 2GTP 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.

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 k cat 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% (k cat 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 k cat 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 (k cat 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 k cat 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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Introduction Previous studies have shown higher breast cancer incidence and mortality among Japanese Brazilians than Japanese. To clarify the difference in hormone levels among populations, we compared postmenopausal endogenous sex hormone levels among Japanese living in Japan, Japanese Brazilians living in São Paulo, and non-Japanese Brazilians living in São Paulo. Methods A cross-sectional study was conducted using a control group of case-control studies in Nagano, Japan and São Paulo, Brazil. Subjects were postmenopausal women aged over 55 years old who provided blood samples. We measured oestradiol, oestrone, androstenedione, dehydroepiandrosterone sulphate (DHEAS), testosterone and free testosterone by radioimmunoassay, bioavailable oestradiol by the ammonium sulphate precipitation method, and sex-hormone binding globulin (SHBG) by immunoradiometric assay. A total of 363 women were included for the present analyses: 185 Japanese, 44 Japanese Brazilians and 134 non-Japanese Brazilians. Results Japanese Brazilians had significantly higher levels of oestradiol, bioavailable oestradiol, oestrone, testosterone, and free testosterone, and lower SHBG levels than Japanese. Japanese Brazilians also had significantly higher levels of bioavailable oestradiol, oestrone, and DHEAS, and lower levels of SHBG and androstenedione than non-Japanese Brazilians. Levels of oestradiol, testosterone, and free testosterone, however, did not differ between Japanese Brazilians and non-Japanese Brazilians. These differences were observed even after adjustment for known breast cancer risk factors. Conclusions We found higher levels of oestrogens and androgens in Japanese Brazilians than in Japanese, and similar to or higher levels than in non-Japanese Brazilians. Our findings may help explain the increase in incidence and mortality of breast cancer among Japanese Brazilians. P1-447 HOW LONG DO PATIENTS IN THE UK GET TREATED FOR NON-SPECIFIC RESPIRATORY SYMPTOMS BY GENERAL PRACTITIONERS BEFORE THEY ARE DIAGNOSED WITH LUNG CANCER?doi:10.1136/jech.2011.142976g.37 B Iyen-Omofoman,* L Tata, R Hubbard. University of Nottingham, Nottingham, UK Introduction Most people with lung cancer in the UK are diagnosed late when curative treatment is no longer an option. This research aimed to determine the pattern of symptom reporting to GPs before lung cancer diagnosis to establish whether there is potential for developing a scoring system to allow cases to be diagnosed earlier.Methods We used data from The Health Improvement Network (THIN)dA computerised longitudinal primary care database. We identified 12 121 incident cases of lung cancer diagnosed between 2000 and 2009 and matched each case with up to four controls by age, sex and general practice (n¼48 216). Conditional logistic regression was carried out to estimate the ORs for symptoms 2 years before and 1 year before lung cancer diagnosis in cases compared to controls. Results A sharp increase in the consultation frequency among lung cancer cases was found for cough, haemoptysis, chest/shoulde...