Catalytic Mechanism for the Conversion of Salicylate Into Catechol by the Flavin-Dependent Monooxygenase Salicylate Hydroxylase

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is \u3e105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis

The electronic origin of unusually large nJFN coupling constants in some Fluoroximes

SOPPA(CCSD) calculations show that the FC term is the most important contribution to the through-space transmission of JFN coupling constants for the fluoroximes studied in this work. Because of the well-known behavior of FC term, a new rationalization for the experimental TSJFN SSCC is presented. It is mainly based on the overlap matrix (Sij) between fluorine and nitrogen lone pairs obtained from NBO analyses. An expression is proposed to take into account the influence of the electronic density (Dij) between coupled nuclei as well as the s% character at the site of the coupling nuclei of bonds and non-bonding electron pairs involved in Dij. In using this approach, a linear correlation between TSJFN versus Dij is obtained. The most important aspect of this rationalization is related to the facility for understanding the behavior of some unusual experimental coupling constants. It is shown that, at least in this case, the electronic origin of the so-called through-space coupling is transmitted through to the overlap of orbitals on the coupled atoms, suggesting that, at least for these compounds, instead of through-space coupling, it should better be dubbed as ‘through overlapping orbital coupling’.Fil: Favaro, Denize C.. Universidade Estadual de Campinas; BrasilFil: Contreras, Ruben Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Tormena, Claudio F.. Universidade Estadual de Campinas; Brasi

Unusual Through-Space, TS, Pathway for the Transmission of JFHf Coupling: 2‑Fluorobenzaldehyde Study Case

The 1 H NMR spectra of the title compound in nonpolar and polar solvents and theoretical calculation of spin−spin coupling constants (SSCCs) show that TSJFHf SSCC, where TS stands for “through-space”, in polar solvents is amenable to measurement only in the trans conformer. The mechanisms for transmission pathways to such unusual SSCCs are rationalized in terms of the molecular electronic structure. It is stressed that such a result calls for some caution when intending to use TSJFH as a probe to detect the spatial proximity between fluorine and hydrogen atoms.Fil: Favaro, Denize C.. Universidad de Campinas. Instituto de Química; Brasil;Fil: Contreras, Ruben Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina; Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina;Fil: Tormena, Cláudio F.. Universidad de Campinas. Instituto de Química; Brasil

Stereochemical dependence of 3J CH coupling constants in 2-substituted 4-t-butyl-cyclohexanone and their alcohol derivatives

Theoretical and experimental studies on 3J C2H6eq NMR spin - spin coupling constants in both the 2-X-4-t-butyl-cyclohexanone (X = H, CH 3, F, Cl, and Br) and in their alcohol derivatives series are reported. Results thus found are rationalized in terms of the transmission of the Fermi contact contribution to such couplings. To this end, dependencies of 3J C2H6eq couplings versus the C 2 - C 1 - C 6 angle are compared in both series for equatorial and axial X orientations. The main trend is described in terms of the rear lobes interaction. Besides, for X = halogen atom in equatorial orientation a rather strong interaction between oxygen and halogen lone pairs is observed, and its influence on 3J C2H6eq couplings is discussed and rationalized in terms of different Fermi contact transmission pathways. (Figure presented) © 2011 American Chemical Society.Fil: Favaro, Denize C.. Universidade Estadual de Campinas; BrasilFil: Ducati, Lucas C.. Universidade Estadual de Campinas; BrasilFil: Dos Santos, Francisco P.. Universidade Estadual de Campinas; BrasilFil: Contreras, Ruben Horacio. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Tormena, Claudio F.. Universidade Estadual de Campinas; Brasi

The Conformational Analysis Of 2-halocyclooctanones.

The establishment of the most stable structures of eight membered rings is a challenging task to the field of conformational analysis. In this work, a series of 2-halocyclooctanones were synthesized (including fluorine, chlorine, bromine and iodine derivatives) and submitted to conformational studies using a combination of theoretical calculation and infrared spectroscopy. For each compound, four conformations were identified as the most important ones. These conformations are derived from the chair-boat conformation of cyclooctanone. The pseudo-equatorial (with respect to the halogen) conformer is preferred in vacuum and in low polarity solvents for chlorine, bromine and iodine derivatives. For 2-fluorocyclooctanone, the preferred conformation in vacuum is pseudo-axial. In acetonitrile, the pseudo-axial conformer becomes the most stable for the chlorine derivative. According to NBO calculations, the conformational preference is not dictated by electron delocalization, but by classical electrostatic repulsions.137176-8

Importance of the β5−β6 Loop for the structure, catalytic efficiency and stability of carbapenem-hydrolyzing class D β-Lactamase subfamily OXA-143

The class D beta-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower K-m) and a higher turnover number (higher k(cat)). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase583436043616CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIGFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP438316/2018-5não temAPQ:90941142011/0777-5; 2017/17303-7; 2017/22822-3The authors thank the facilities NMR Laboratory, Chemistry Institute IQ-UNICAMP, Núcleo de Computação Científica da Universidade do Estado de São Paulo (NCC/GridUNESP), and Centro Nacional de Processamento de Alto Desempenho em São Paulo (CENAPAD-SP) for computational resources, The authors also thank the Chemistry Institute of the University of Campinas and the Chemistry Institute of the University of São Paulo. Ronaldo Nagem and Tiago Brandão are acknowledged for their financial support during time spent at UFMG. The authors finally thank Stefanya Velázques Gómez for helpful discussions, Cláudio F. Tormena and Carlos Ramos for kindly helping with reagents and equipment, and Annelize Z. B. Aragão for support with mutations and protein purificatio

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from paracoccidioides brasiliensis

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coil BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an alpha beta alpha beta alpha beta topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (H-1-N-15 HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies112151162CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ484466/2007-0; 457851/2014-7This work was supported by the Laboratório Nacional de Luz Síncrotron (LNLS – Brazilian Synchrotron Light Laboratory, Projects MX1-13949 and MX2-18603) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Projects 484466/2007-0 and 457851/2014-7). We would also like to acknowledge the support and collaboration of Dra. Terese Bergfors during the preliminary rPb27 crystallization attempt

Uncovering the Association Mechanism between Two Intrinsically Flexible Proteins

The understanding of protein–protein interaction mechanisms is key to the atomistic description of cell signaling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Nt behaves as a random coil, while VirB9Ct is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence, and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Nt coupled folding and binding mechanism. Qualitative data analysis suggested that VirB9Ct preferentially binds to VirB7Nt by way of a conformational selection mechanism at lower temperatures. However, at higher temperatures, energy barriers between different VirB9Ct conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may provide alternative pathways toward the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state

Effect of Counterions on the Shape, Hydration, and Degree of Order at the Interface of Cationic Micelles: the Triflate Case

Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA(+) micelles. Above a defined concentration, sodium triflate (NaTf induces a phase separation in dodecyltrimethylammonium triflate (DTATf micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. the structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the -CF3 group in Tf- was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles.GENFIT programFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia de Fluidos Complexos (INCT-FCx)Nucleo de Apoio a Pesquisa de Fluidos Complexos (NAP-FCx)Univ São Paulo, Inst Quim, São Paulo, BrazilUniv São Paulo, Inst Fis, BR-01498 São Paulo, BrazilUniv Regensburg, Inst Phys & Theoret Chem, D-93053 Regensburg, GermanyUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, Santo Andre, BrazilUniv São Paulo, FFCLRP, Dept Quim, BR-14049 Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFAPESP: 2007/50970-5FAPESP: 2008/50041-7Web of Scienc

Effect of Counterions on the Shape, Hydration, and Degree of Order at the Interface of Cationic Micelles: The Triflate Case

Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA⁺ micelles. Above a defined concentration, sodium triflate (NaTf) induces a phase separation in dodecyltrimethylammonium triflate (DTATf) micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. The structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the −CF₃ group in Tf^– was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles