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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 A 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 >105 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 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 Pseudomonas putida 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 kcat/Km values close to 10(6) 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.

Os Hidrocarbonetos Aromáticos Policíclicos (HAPs), um grupo de compostos orgânicos formados por anéis benzênicos fusionados, são contaminantes ambientais amplamente distribuídos tanto devido a fontes naturais quanto antropogênicas, sendo as atividades de produção de carvão vegetal e mineral, de refino e transporte de petróleo os principais contribuintes para o despejo localizado de HAPs. Devido à sua ocorrência ubíqua , potencial de bioacumulação e atividade carcinogênica, vários HAPs foram listados como poluentes prioritários para a remediação destacando a importância da sua remoção do meio ambiente. Diversos microrganismos têm sido extensamente estudados em virtude de sua versatilidade em degradar uma gama de compostos aromáticos. A bactéria Pseudomonas putida G7 possui um plasmídeo associado ao metabolismo do naftaleno, o mais simples e abundante HAP, cuja degradação ocorre através de duas vias catabólicas ditas superior e inferior. NahI, uma 2- hidroximuconato semialdeído desidrogenase (2-HMSD) atuante na via inferior, converte 2- hidroximuconato semialdeído (2-HMS) a 2-hidroximuconato (2-HM) na presença de NAD+. Mesmo a despeito da baixa identidade de sequencia, uma característica comum entre enzimas aldeído desidrogenases (ALDHs) é a manutenção de um enovelamento estrutural semelhante, salvas as inúmeras particularidades concernentes ao estado de oligomerização e às interações específicas estabelecidas entre enzima, substrato e cofator. Assim, este trabalho se propôs a caracterizar cinética e estruturalmente a enzima NahI, uma vez que, através da elucidação de sua estrutura tridimensional e comparação com as de outras ALDHs, possibilita-se inferir distinções no sítio catalítico responsáveis por mudanças sistemáticas nas propriedades cinéticas destas enzimas sobre uma variedade de substratos aldeídos. Após purificação por cromatografia de afinidade e de exclusão molecular, ensaios de espalhamento dinâmico de luz (DLS) e espalhamento de raios-X a baixo ângulo (SAXS) foram realizados para analisar o estado oligomérico da enzima em solução, identificada como um tetrâmero. No que diz respeito à estrutura cristalográfica, NahI apresentou o enovelamento / típico da superfamília ALDH organizado em domínio de oligomerização e domínios de ligação ao dinucleotídeo e ao substrato, este último também conhecido como catalítico. Através de operações de simetria cristalográfica, observou-se o tetrâmero de NahI formado por um dímero de dímeros. Ensaios de cinética confirmaram a preferência da enzima para o substrato alifático 2-HMS, porém não se observou qualquer atividade sobre salicilaldeído, substrato aromático natural de NahF, uma ALDH pertencente à mesma via de degradação do naftaleno. Mutações foram propostas para NahI a fim de aumentar o volume do bolso catalítico, uma vez que resíduos de cadeia lateral volumosa poderiam dificultar a atividade desta enzima sobre substratos aromáticos. Surpreendentemente, as formas mutantes não apresentaram atividade sobre salicilaldeído. Além disso, o alargamento da entrada do bolso catalítico afetou ligeiramente a afinidade da enzima para o seu substrato natural, 2-HMS. Sugere-se ainda que o resíduo L156 parece ser crucial para a oxidação do substrato. Os resultados desta investigação apresentam, pela primeira vez, características estruturais e cinéticas em relação à enzima NahI e favorecem futuros estudos sobre a identidade e função de resíduos cruciais para a catálise e a descrição de como a reação se processa. The Polycyclic Aromatic Hydrocarbons (PAHs), a group of organic compounds consisting of fused benzene rings, are contaminants widely distributed in the environment due to natural as well as anthropogenic sources. Activities of production of coal, refining and transportation of oil are the major contributors to the contamination with PAHs. Due to their ubiquitous occurrence, potential to bio-accumulate and carcinogenic activity, a number of PAHs were listed as priority pollutants for remediation highlighting the importance of their removal from the environment. Several microorganisms have been extensively studied because of its versatility to degrade a wide range of aromatic compounds. The bacterium Pseudomonas putida G7 has a plasmid associated with the metabolism of naphthalene, the simplest and most abundant PAH. Its degradation by P. putida occurs through the upper and the lower catabolic pathways. NahI, a 2-hydroxymuconate semialdehyde dehydrogenase (2-HMSD) belonging to the lower pathway, converts 2-hydroxymuconate semialdehyde (2-HMS) to 2- hydroxymuconate (2-HM) in the presence of NAD+. A common feature among aldehyde dehydrogenases (ALDHs) is the similar scaffold, even despite their overall low sequence identity, modes of oligomerization and substrate specificity. Thus, this study was proposed to characterize the structure and kinetics of NahI. By elucidation of its three-dimensional structure and comparison with other ALDHS, it becomes possible to infer distinctions in the catalytic site that are responsible for systematic changes in the kinetic properties of these enzymes within a variety of aldehyde substrates After purification by affinity and sizeexclusion chromatography, dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) experiments were conducted to analyze the oligomeric state of the enzyme in solution, which was identified as being a tetramer. With regard to the crystal structure, NahI displayed a typical / aldehyde dehydrogenase superfamily fold with three well defined domains: the oligomerization domain, the nucleotide binding and the catalytic domains. Through crystallographic symmetry operations, the NahI tetramer formed by a dimer of dimers was observed. Kinetic assays confirmed the preference of the enzyme for the aliphatic substrate 2-HMS. On the other hand, NahI showed no activity with salicylaldehyde, the natural substrate for NahF enzyme, another ALDH belonging to the same naphthalenedegradation pathway. Mutations have been proposed for NahI in order to increase the volume of the catalytic pocket, since bulky side chain residues could hinder the activity of this enzyme with aromatic substrates. Surprisingly, the mutant forms showed no activity with salicylaldehyde. Moreover, the enlargement of the catalytic pocket entrance slightly affected the affinity of the enzyme for its natural substrate, 2-HMS. It also suggests that the L156 residue appears to be crucial for the oxidation of the substrate. For the first time, these findings show the structural and kinetic properties relative to the NahI enzyme and highlights further studies concerning the identity and function of critical residues for catalysis and description of how the reaction proceeds.

The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase required for conversion of 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD+. NahI is in one family of the NAD(P)+-dependent aldehyde dehydrogenase superfamily (ALDH8). In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress at 12 ºC as an N-terminal hexa-histidine-tagged fusion protein (6xHis-NahI). After the soluble protein was purified by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were also evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted for the recombinant enzyme and, for the first time, KM (1.3 ± 0.3 μM) and kcat (0.9 s−1) values were determined for this enzyme (at presumed NAD+ saturation). NahI is highly specific for its biological substrate (2-hydroxymuconate semialdehyde) and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway.

A família gênica masp (proteína de superfície associada a mucina), identificada durante a anotação do genoma do Trypanosoma cruzi, é a segunda maior família mutligênica neste parasito, consistindo de aproximadamente 1400 membros. Membros da família MASP contêm domínios N- e C-terminais conservados que codificam para um peptídeo sinal e um sítio de adição de âncora de GPI, respectivamente. A região central é variável em comprimento e em sequência de aminoácidos, e contém um grande repertório de motivos repetitivos, os quais sugerem que MASP esteja envolvida em interações parasito-hospedeiro. Uma vez que nenhum membro da família MASP foi caracterizado até o momento, decidiu-se estudar as estruturas secundária e terciária de diferentes MASPs. Para este objetivo, optou-se por clonar e expressar em Escherichia coli a região central das sequências de quatro membros MASP. Para isso, algumas características das proteínas alvo foram consideradas, tais como curto comprimento da sequência de aminoácidos, em torno de 170 resíduos, baixo conteúdo de sítios potenciais de glicosilação e baixo grau de desordem estrutural. Uma vez que regiões de baixa complexidade normalmente correspondem a segmentos desenovelados ou estruturas extendidas, foi utilizado o servidor PONDR VL-XT para predizer regiões desordenadas nos quatro membros MASP. Proteínas nativamente desenoveladas têm sido descritas por possuírem elevada taxa de resíduos carregados e baixa taxa de resíduos hidrofóbicos. Gráficos de carga líquida média versus taxa de hidrofobicidade foram obtidos para as regiões centrais das quatro MASPs. Estas análises revelaram a presença das quatro regiões centrais no espaço designado para proteínas nativamente desenoveladas. As sequências foram amplificadas por PCR, clonadas nos sistemas de clonagem TOPO e/ou pGEM-T e submetidas ao sequenciamento de DNA. Posteriormente, os produtos de PCR foram transferidos para dois vetores de expressão derivados do vetor pET-28a (Novagen), que foram modificados no Centro de Biologia Molecular e Estrutural (CeBiME, Campinas / SP). Em ambos os vetores, o sítio de reconhecimento por trombina foi substituído pelo sítio de reconhecimento da protease TEV (tobacco etch vírus). As quatro proteínas MASP em fusão a cauda de histidina N-terminal foram expressas na forma solúvel, enquanto que aquelas fusionadas a GST N-terminal foram expressas como corpos de inclusão insolúveis, exceto a recombinante MASP1:GST. A purificação da MASP 3 fusionada a cauda de histidina foi realizada por cromatografia de afinidade. Após a clivagem da etiqueta por TEV, a proteína foi purificada por cromatografia de troca iônica. O estudo da estrutura tridimensional de proteínas MASP permitirá uma compreensão mais detalhada destas moléculas, tal como a localização de seus epítopos e seu enovelamento, o que pode contribuir para a identificação de sua função biológica no T. cruzi. The Trypanosoma cruzi masp (mucin associated surface protein) gene family was identified during the annotation of the genome as the second largest gene family in this human pathogen, consisting of approximately 1400 members. MASP members contain N- and C- terminal conserved domains that encode a putative signal peptide and a GPI-anchor addition site, respectively. The central region is variable both in length and in amino acid sequence and contains a large repertoire of repetitive motifs, which suggests MASP may be involved in parasite-host cell interaction. Since no member of the MASP family has been characterized to date, we have decided to study the secondary and tertiary structures of distinct MASPs. For this purpose, we have decided to clone and express in E. coli the central region sequences of four MASP members. For this, some features of the target proteins were considered such as short amino acid sequence length, around 170 residues, low content of potential glycosylation sites and low intrinsic disorder degree. Knowing that sequence regions with low complexity nearly always correspond to nonfolding segments or extended structures, we have used the web server PONDR VL-XT to predict disordered regions in MASPs. Natively unfolded proteins have been described to possess a high ratio of charged residues and a low ratio of hydrophobic residues. The mean net charge versus mean hydrophobicity ratio was ploted for the four MASPs central region. This analysis showed that the four central regions falls in the phase space of natively unfolded proteins. After that, the sequences were PCR amplified and cloned into TOPO and/or pGEM-T cloning systems and submitted to DNA sequencing. Subsequently, the PCR products were transferred to two expression vectors derived from pET-28a vector (Novagen), which were modified in the Center of Molecular and Structural Biology (CeBiME, Campinas/SP). In both vectors, the thrombin recognition site was replaced by a TEV (tobacco etch virus) protease recognition site. The four N-terminal fusion His-tag MASP proteins were expressed in the soluble forms, whereas the N-terminal GST fusion MASPs were expressed as insoluble inclusion bodies, except the recombinant GST:MASP1. Purification of the Histidine-tagged MASP3 was carried out by affinity chromatography. After His-tag cleavage by TEV, the protein was purified by ion exchange chromatography. The study of the three-dimensional structure of MASP proteins will enable a more detailed understanding of these molecules, such as the location of their epitopes and its fold, which may contribute to the identification of its biological function on T. cruzi.

Polycyclic aromatic hydrocarbons (PAHs) are a group of aromatic hydrocarbons composed of two or more fused rings and include naphthalene, phenanthrene, pyrene and benzo[a]pyrene amongst others. Exposure to naphthalene has been associated with several toxic manifestations in humans and laboratory animals, with the lens of the eye and the lungs being most sensitive [1]. Additionally, this compound has been reclassified as a possible human carcinogen by the US Environmental Protection Agency and the International Agency for Research on Cancer. It has recently been suggested that naphthalene undergoes metabolic activation to 1,2-naphthoquinone, which reacts with DNA, leading to the formation of apurinic sites on DNA and depurinating DNA adducts [2]. Moreover, a number of studies have attested to the carcinogenic and mutagenic properties of more complex PAHs, suggesting the need for further work on the elimination of these compounds from the environment. The Gram-negative bacterium P. putida G7 is among the best studied naphthalene-degrading species. The genes associated with naphthalene metabolism are localized on NAH7, an 83 kilobase plasmid. In P. putida G7 the naphthalene-oxidation genes are organized into two operons under salicylate control. The first operon (upper naphthalene-degradation pathway) includes the genes nahAaAbAcAdBCDEF, which code for the conversion of naphthalene to salicylate, while the second operon (lower pathway) includes the genes nahGTHINLOMKJ responsible for the oxidation of salicylate via the catechol meta-cleavage pathway [3]. In the last years, our group focused on the structure elucidation and the kinetic characterization of the P. putida G7 enzymes involved in the naphthalene degradation pathway. We intend to present these results which basically describe the 3D structures of NahB, NahF, NahG, NahI, NahK and NahK/NahL complex and some kinetic data of these enzymes and their mutants. This work was supported by FAPEMIG, CNPq and VALE S.A.