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

Um estudo teórico de propriedades moleculares em complexos de hidrogênio trimoleculares C2H4···2HF, C2H2···2HF e C3h6···2HF A theoretical study of molecular properties of C2H4···2HF, C2H2···2HF AND C3H6···2HF trimolecular hydrogen-bonded complexes

<abstract language="eng">We present a theoretical study of molecular properties in C2H4···2HF, C2H2···2HF and C3H6···2HF trimolecular hydrogen-bonded complexes. From B3LYP/6-311++G(d,p) calculations, the most important structural deformations are related to the C=C (C2H4), C&#8801;C (C2H2), C-C (C3H6) and HF bond lengths. According to the Bader's atoms in molecules and CHELPG calculations, it was identified a tertiary interaction between the fluorine atom of the second hydrofluoric acid molecule and hydrogen atoms of the ethylene and acetylene within the C2H4···2HF and C2H2···2HF complexes, respectively. Additionally, the evaluation of the infrared spectrum characterized the new vibrational modes and bathochromic effect of the HF molecules

Mechanistic Aspects of Phosphate Diester Cleavage Assisted by Imidazole. A Template Reaction for Obtaining Aryl Phosphoimidazoles

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of <i>o</i>-(2′-imidazolyl)­naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a S_N2­(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the <i>p</i>-nitrophenolate leaving group. The P–O bond to the leaving group is about 50–60% broken in the transition state

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD⁺ as a cofactor, the last reaction of the upper degradation pathway of naphthalene in <i>Pseudomonas putida</i> G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large <i>k</i>_cat/<i>K</i>_m values close to 10⁶ M^–1 s^–1. The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes

Estado e mercado: adversários ou aliados no processo de implementação da Política Nacional de Alimentação e Nutrição? Elementos para um debate sobre medidas de regulamentação State and market: adversaries or allies in the implementation of the National Food and Nutrition Policy? Some reflections on regulation measures

A alimentação e a nutrição constituem requisitos básicos para a promoção e a proteção da saúde, possibilitando a afirmação plena do potencial de crescimento e desenvolvimento humano, com qualidade de vida e cidadania. As mudanças na relação do Estado, sociedade civil e mercado têm trazido uma série de conseqüências sociais adversas, especialmente no campo da saúde e nutrição. Este artigo tem como propósito provocar algumas reflexões relativas à necessidade de pactuação entre Estado, sociedade e mercado para a efetivação de medidas capazes de atuar positivamente no controle da obesidade e conseqüentemente melhorar as condições de saúde e nutrição da população brasileira. Apresenta uma breve descrição de contexto histórico sobre política social no Brasil e seus reflexos específicos na temática de alimentação e nutrição; identifica aspectos políticos principais para uma análise das diretrizes da Política Nacional de Alimentação e Nutrição; destaca o fortalecimento de medidas de regulamentação como estratégia de enfrentamento do problema de excesso de peso / obesidade na sociedade, identificando suas raízes a partir do modo de viver urbano, globalizado e neoliberal das ultimas décadas, e, por fim, sugere elementos para enriquecer esse debate. Medidas de regulamentação parecem poder influenciar positivamente nas ações de promoção da saúde, pois estabelecem limites e um conjunto de critérios para mediar as relações entre sociedade e mercado, além de refletir o apoio e a proteção da saúde do cidadão como objetivo principal do Estado nesse processo.<br>Food and nutrition constitute basic requirements for health promotion and protection, enabling the full expression of the human potential for growth and development, with quality of life and citizenship. Changes in the relationship between State, Civil Society, and the Market have brought several adverse social consequences, particularly in the health and nutrition field. This article intends to stimulate some reflections related to the need of an agreement between State, Civil Society and the Market for the accomplishment of measures that are able to contribute to obesity control and, as a consequence, to improve health and nutrition conditions of the Brazilian population. The article also presents a brief description of the historical context of social policies in Brazil and their specific consequences to food and nutrition matters; it identifies the main political aspects to analyze guidelines of the National Food and Nutrition Policy; it emphasizes the strengthening of regulation measures as a strategy to face the problem of excess of weight / obesity in society, identifying its roots in the urban, 'global' and neoliberal way of life of the last decades and, as a conclusion, it suggests more elements to enrich this debate. Regulation Measures seem to have a positive influence on health promotion actions by setting up the boundaries and criteria to mediate the relationship between Civil Society and the Market, besides reflecting the support and protection of the citizen's health as the main purpose of the State in this process

Síntese de proteína microbiana e concentrações de uréia em vacas alimentadas com diferentes fontes de proteína Estimation of microbial protein synthesis and urea nitrogen metabolism in lactating dairy cows fed diets supplemented with different protein sources

Foram utilizadas 12 vacas Holandesas puras e mestiças, distribuídas em três quadrados latinos 4 x 4, organizados de acordo com os dias em lactação, com o objetivo de avaliar o efeito de diferentes fontes protéicas sobre a síntese, a eficiência de síntese de proteína microbiana, a concentração de nitrogênio uréico no soro (NUS) e no leite (NUL), a concentração de nitrogênio amoniacal e o pH ruminal. Utilizou-se silagem de milho como volumoso, na proporção de 60% da MS total. Os concentrados foram constituídos de diferentes fontes protéicas (FS - farelo de soja; FA38 - farelo de algodão 38%PB; FA28 - farelo de algodão 28%PB e FSU - farelo de soja + 5% de uréia/sulfato de amônia na MS do concentrado). As coletas spot de urina e de sangue foram realizadas no 18º dia do período experimental 4 horas após o fornecimento da alimentação aos animais, no período da manhã. Não foram observadas diferenças entre as dietas para o volume urinário (V), a excreção total de derivados de purinas (PT), a síntese e a eficiência de síntese de PB microbiana, expressa em g de PB/kg de NDT consumido. As concentrações de NUS e NUL também não diferiram entre as dietas. As concentrações de NUS e NUL e a síntese de PB microbiana não foram influenciadas pelas diferentes fontes de proteína dietéticas, inclusive com a adição de uréia (5% MS do concentrado).<br>Twelve Holstein lactating dairy cows were blocked by days in milk and randomly assigned to three replicated 4 x 4 Latin square to evaluate the effect of different protein sources on efficiency of microbial protein synthesis, concentration of serum (NUS) and milk (MUN) urea nitrogen, and ruminal metabolism. A basal corn silage diet (60% of the total dry matter) was fed plus one of the following proteins sources (DM basis): soybean meal (SBM), cottonseed meal with 38% of crude protein (CSM38), cottonseed meal with 28% of crude protein (CSM28), or soybean meal plus 5% of urea/ammonium sulfate (SBMU). Each experimental period lasted 18 days with 11 days for diet adaptation and seven days for sample collection. Samples of urine and blood were obtained approximately four hours after the morning feeding on day 18 of each period. No significant differences among diets were observed for urinary volume (V), total purine derivatives excretion (PT), microbial CP synthesis and microbial efficiency, expressed in g of CP/kg of TDN intake. In addition, concentration of both NUS and MUN also did not differ among diets. It can be concluded that NUS and MUN as well as microbial CP synthesis and efficiency were not affected by feeding different protein sources to lactating dairy cows