RESUMO
We report the synthesis of a new asymmetric heptadentate ligand based on the 1,3-diaminopropan-2-ol backbone. The ligand 3-[[3-(bis-pyridin-2-ylmethyl-amino)-2-hydroxy-propyl]-(2-carbamoyl-ethyl)-amino]-propionamide (HL1) contains two amide and two pyridine groups attached to the 1,3-diaminopropan-2-ol core. Reaction between HL1 and Zn(ClO4)2.6H2O resulted in the formation of the dinuclear [Zn2(L1)(µ-OAc)](ClO4)2 complex, characterized by single crystal X-ray diffraction, 1H, 13C and 15N NMR, ESI-(+)-MS, CHN elemental analysis as well as infrared spectroscopy. The phosphatase activity of the complex was studied in the pH range 6-11 employing pyridinium bis(2,4-dinitrophenyl)phosphate (py(BDNPP)) as substrate. The complex exhibited activity dependent on the pH, presenting an asymmetric bell shape profile with the highest activity at pH 9; at high pH ligand exchange is rate-limiting. The hydrolysis of BDNPP- at pH 9 displayed behavior characteristic of Michaelis-Menten kinetics, with kcat = 5.06 × 10-3 min-1 and Km = 5.7 ± 1.0 mM. DFT calculations map out plausible reaction pathways and identify a terminal, Zn(II)-bound hydroxide as likely nucleophile.
Assuntos
Monoéster Fosfórico Hidrolases , Zinco , Zinco/química , Ligantes , Hidrólise , Cinética , Monoéster Fosfórico Hidrolases/química , Cristalografia por Raios XRESUMO
Pseudomonas aeruginosa is a highly pathogenic Gram-negative microorganism associated with high mortality levels in burned or immunosuppressed patients or individuals affected by cystic fibrosis. Studies support a colonization mechanism whereby P. aeruginosa can breakdown the host cell membrane phospholipids through the sequential action of two enzymes: (I) hemolytic phospholipase C acting upon phosphatidylcholine or sphingomyelin to produce phosphorylcholine (Pcho) and (II) phosphorylcholine phosphatase (PchP) that hydrolyzes Pcho to generate choline and inorganic phosphate. This coordinated action provides the bacteria with carbon, nitrogen, and inorganic phosphate to support growth. Furthermore, PchP exhibits a distinctive inhibition mechanism by high substrate concentration. Here, we combine kinetic assays and computational approaches such as molecular docking, molecular dynamics, and free-energy calculations to describe the inhibitory site of PchP, which shares specific residues with the enzyme's active site. Our study provides insights into a coupled inhibition mechanism by the substrate, allowing us to postulate that the integrity of the inhibition site is needed to the correct functioning of the active site. Our results allow us to gain a better understanding of PchP function and provide the basis for a rational drug design that might contribute to the treatment of infections caused by this important opportunistic pathogen.
Assuntos
Monoéster Fosfórico Hidrolases , Pseudomonas aeruginosa , Humanos , Simulação de Acoplamento Molecular , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/química , Fosforilcolina/química , Fosforilcolina/farmacologia , Pseudomonas aeruginosa/metabolismoRESUMO
Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.
Assuntos
Entamoeba histolytica/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , 6-Fitase/metabolismo , Sítios de Ligação , Domínio Catalítico , Difosfatos/metabolismo , Entamoeba histolytica/genética , Humanos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Inositol polyphosphate 5-phosphatase (OCRL-1) participates in the regulation of multiple cellular processes, through the conversion of phosphatidylinositol 4,5-phosphate to phosphatidylinositol 4-phosphate. Mutations in this protein are related to Lowe syndrome (LS) and Dent-2 disease. In this study, the impact of Lowe syndrome mutations on the interactions of OCRL-1 with other proteins was evaluated through bioinformatic and computational approaches. In the functional analysis of the interaction network of the proteins, we found that the terms of gene ontology (GO) of greater significance were related to the intracellular transport of proteins, the signal transduction mediated by small G proteins and vesicles associated with the Golgi apparatus. From the proteins present in the GO terms of greater significance Rab8a was selected because its interaction facilitates the intracellular distribution of OCRL-1. The mutation p.Asn591Lys, present in the interaction domain of OCRL-1 and Rab8a, was studied using molecular dynamics. The molecular dynamics analysis showed that the presence of this mutation causes changes in the positional fluctuations of the amino acids and affects the flexibility of the protein making the interaction with Rab8a weaker. Rab proteins establish some specific interactions, which are important for the intracellular localization of OCRL-1; therefore, our findings suggest that the phenotype observed in patients with LS, in this case, is due to the destabilizing effect of p.Asn591Lys affecting the localization of OCRL-1 and indirectly its 5-phosphatase activity in the Golgi apparatus, endosomes, and cilia.
Assuntos
Simulação de Dinâmica Molecular , Mutação , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Mapeamento de Interação de Proteínas , Substituição de Aminoácidos , Ligação de Hidrogênio , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Termodinâmica , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.
Assuntos
Biodegradação Ambiental , Agentes Neurotóxicos/metabolismo , Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Agentes Neurotóxicos/química , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Teoria Quântica , Sarina/química , Sarina/metabolismoRESUMO
Ostreococcus tauri, the smallest free-living (non-symbiotic) eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP), homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.
Assuntos
Clorófitas/enzimologia , Glucanos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Dimerização , Cinética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The structural and functional properties of zinc(ii) complexes of two nitrogen rich polydentate ligands, HTPDP = 1,3-bis(bis-pyridin-2-ylmethylamino)propan-2-ol and HTPPNOL = N,N,N'-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol, are compared. HTPDP is a hepta-dentate ligand with four pyridyl groups attached to a 1,3-diaminopropan-2-ol backbone while HTPPNOL contains only three pyridyl groups. In reactions with Zn(ClO4)2, HTPDP forms a dinuclear zinc compound [Zn2(TPDP)(OAc)](ClO4)2, 1. On the other hand, mononuclear [Zn(HTPPNOL)](ClO4)2, 2, and tetranuclear [Zn4(TPPNOL)2(OAc)3](ClO4)3, 3, complexes were isolated with the ligand HTPPNOL. Kinetic measurements with the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) revealed that compound 1 (kcat = 31.4 × 10-3 min-1) is more reactive than 3 (kcat = 7.7 × 10-3 min-1) at pH = 8.5, whilst the mononuclear compound 2 is inactive. Compound 1 displays a typical steady-state kinetic behaviour, while compound 3 exhibits steady-state behaviour only â¼120 s after starting the reaction, preceded by a burst-phase. 31P NMR studies confirm that 1 can promote the hydrolysis of both ester bonds in BDNPP, generating the monoester DNPP and inorganic phosphate in the process. In contrast, DNPP is not a substrate for 3. The crystal structure of the complex formed by 3 and DNPP reveals the formation of a tetranuclear zinc complex [Zn4(TPPNOL)2(DNPP)2](ClO4)2, 4, in which the phosphate moiety of DNPP adopts an unusual tridentate µ-η1:η1:η1 coordination mode.
Assuntos
Materiais Biomiméticos/química , Complexos de Coordenação/química , Organofosfatos/química , Monoéster Fosfórico Hidrolases/química , Zinco/química , Biomimética , Catálise , Cristalografia por Raios X , Hidrólise , Cinética , Ligantes , Modelos Moleculares , Propanóis/química , Piridinas/químicaRESUMO
The TPM domain constitutes a family of recently characterized protein domains that are present in most living organisms. Although some progress has been made in understanding the cellular role of TPM-containing proteins, the relationship between structure and function is not clear yet. We have recently solved the solution and crystal structure of one TPM domain (BA42) from the Antarctic bacterium Bizionia argentinensis. In this work, we demonstrate that BA42 has phosphoric-monoester hydrolase activity. The activity of BA42 is strictly dependent on the binding of divalent metals and retains nearly 70% of the maximum at 4 °C, a typical characteristic of cold-adapted enzymes. From HSQC, 15 N relaxation measurements, and molecular dynamics studies, we determine that the flexibility of the crossing loops was associated to the protein activity. Thermal unfolding experiments showed that the local increment in flexibility of Mg2+ -bound BA42, when compared with Ca2+ -bound BA42, is associated to a decrease in global protein stability. Finally, through mutagenesis experiments, we unambiguously demonstrate that the region comprising the metal-binding site participates in the catalytic mechanism. The results shown here contribute to the understanding of the relationship between structure and function of this new family of TPM domains providing important cues on the regulatory role of Mg2+ and Ca2+ and the molecular mechanism underlying enzyme activity at low temperatures.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Flavobacteriaceae/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Estabilidade Enzimática , Flavobacteriaceae/genética , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Metais/metabolismo , Modelos Moleculares , Mutação , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG()=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH()=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH()=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP.