RESUMO
The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent ß-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous ß-keto acid decarboxylase and the first crystal structure of a 4-OD.
Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Cetoácidos/química , Magnésio/química , Pseudomonas putida/química , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Cristalografia por Raios X , Descarboxilação , Cetoácidos/metabolismo , Magnésio/metabolismo , Domínios Proteicos , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMO
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/ß 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.
Assuntos
Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/química , Cristalografia por Raios X , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Especificidade por Substrato , TemperaturaRESUMO
Since the emergence of the new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) at the end of December 2019 in China, and with the urge of the coronavirus disease 2019 (COVID-19) pandemic, there have been huge efforts of many research teams and governmental institutions worldwide to mitigate the current scenario. Reaching more than 1,377,000 deaths in the world and still with a growing number of infections, SARS-CoV-2 remains a critical issue for global health and economic systems, with an urgency for available therapeutic options. In this scenario, as drug repurposing and discovery remains a challenge, computer-aided drug design (CADD) approaches, including machine learning (ML) techniques, can be useful tools to the design and discovery of novel potential antiviral inhibitors against SARS-CoV-2. In this work, we describe and review the current knowledge on this virus and the pandemic, the latest strategies and computational approaches applied to search for treatment options, as well as the challenges to overcome COVID-19.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Desenho de Fármacos , Descoberta de Drogas/métodos , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Inteligência Artificial , COVID-19/metabolismo , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2/fisiologiaRESUMO
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 2.0â¯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 >105â¯M-1â¯s-1 at pHâ¯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.