Papers by Diogenes santos silva Santos
As doenças infecto-contagiosas, entre elas a tuberculose, estão ressurgindo e cada vez mais sendo... more As doenças infecto-contagiosas, entre elas a tuberculose, estão ressurgindo e cada vez mais sendo causadas por cepas resistentes. Portanto, torna-se fundamental a busca de novos antimicrobianos capazes de atacar alvos exclusivos a estes microorganismos. Um destes alvos, é a rota anabólica do ácido chiquímico utilizada por plantas e bactérias para a síntese dos aminoácidos aromáticos. Recentemente, com a publicação da sequência do Mycobacterium tuberculosis H37Rv, verificou-se a presença dos sete genes codificantes envolvidos nesta via. Um deles, o aroG, codifica a enzima DAPH (3-deoxi-D-arabino-heptulosonato-7-fosfato) Sintase que catalisa o primeiro passo da via do ácido chiquímico, condensando fosfoenolpiruvato e eritrose-4-fosfato. Sabendo-se que o aroG está localizado na posição Rv2178c do referido genoma e tem cerca de 1400bp, o objetivo deste trabalho foi a clonagem e seqüenciamento do aroG, a partir de DNA genômico de M.tuberculosis H37Rv. Para tal, amplificou-se este gene via PCR. O fragmento de DNA amplificado foi clonado no vetor pCR®-Blunt, transformado em células TOP10 e, posteriormente, transferido para o vetor de super-expressão pET 23a(+) e transformado em células de E. coli. A inserção do fragmento nos vetores recombinantes foi confirmada pela digestão com enzimas NdeI e BamHI. A identificação do gene clonado foi feita por seqüenciamento de DNA pelo método de Sanger. Com o gene clonado, pretende-se superexpressar a DAPH Sintase, purificandoa e estudando sua cinética enzimática. As perspectivas deste trabalho incluem a proposta de compostos similares em estrutura química ao substrato ou ao estado de transição da reação enzimática, para que estes possam ser testados como inibidores da DAPH Sintase e, possivelmente, como agentes terapêuticos no tratamento da tuberculose (PADCT e FAPERGS).
Genetics and molecular research : GMR, 2007
The rate at which knowledge about genomic sequences and their protein products is produced is inc... more The rate at which knowledge about genomic sequences and their protein products is produced is increasing much faster than the rate of 3-dimensional protein structure determination by experimental methods, such as X-ray diffraction and nuclear magnetic resonance. One of the major challenges in structural bioinformatics is the conversion of genomic sequences into useful information, such as characterization of protein structure and function. Using molecular dynamics (MD) simulations, we predicted the 3-dimensional structure of an artificially designed three- alpha -helix bundle, called A3, from a fully extended initial conformation, based on its amino acid sequence. The MD protocol enabled us to obtain the secondary, in 1.0 ns, as well as the supersecondary and tertiary structures, in 4.0-10.0 ns, of A3, much faster than previously described for a similar protein system. The structure obtained at the end of the 10.0-ns MD simulation was topologically a three-alpha-helix bundle.
RSC Adv., 2015
Glutamate-47 plays a catalytic role in the mode of action of Mycobacterium tuberculosis cytidine ... more Glutamate-47 plays a catalytic role in the mode of action of Mycobacterium tuberculosis cytidine deaminase.
Virchows Archiv, 2011
The E-twenty-six (ETS) family of transcription factors is known to act as positive or negative re... more The E-twenty-six (ETS) family of transcription factors is known to act as positive or negative regulators of the expression of genes that are involved in diverse biological processes, including those that control cellular proliferation, differentiation, hematopoiesis, apoptosis, metastasis, tissue remodeling, and angiogenesis. Identification of target gene promoters of normal and oncogenic transcription factors provides new insights into the regulation of genes that are involved in the control of normal cell growth and differentiation. The aim of the present investigation was to analyze the differential expression of 11 ETS (ELF-3, ESE3, ETS1, ETV3, ETV4, ETV6, NERF, PDEF, PU1, Spi-B, and Spi-C) as potential markers for prognostic of colorectal cancer. A series of paired tissue biopsies consisting of a tumor and a non-affected control sample were harvested from 28 individuals suffering from diagnosed colorectal lesions. Total RNA was isolated from the samples, and after reverse transcription, differential expression of the select ETS was carried out through real-time polymerase chain reaction. Tumor staging as determined by histopathology was carried out to correlate the degree of tumor invasiveness with the expression of the ETS genes. The results demonstrated a different quantitative profile of expression in tumors and normal tissues. ETV4 was significantly upregulated with further increase in the event of lymph node involvement. PDEF and Spi-B presented downregulation, which was more significant when lymph node involvement was present. These findings were supported by immunohistochemistry of tumoral tissues. The results suggest that select ETS may serve as potential markers of colorectal cancer invasiveness and metastasis.
Proteins: Structure, Function, and Bioinformatics, 2008
Protein Expression and Purification, 2006
The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a... more The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.
Protein Expression and Purification, 2006
Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB,... more Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drugresistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.
PLoS ONE, 2013
Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-a... more Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-a-1-pyrophosphate (PRPP) to uridine 59-monophosphate (UMP) and pyrophosphate (PP i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and Nterminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
Monatshefte für Chemie - Chemical Monthly, 2013
The low aqueous solubility of drug-like compounds has been described as one of the main problems ... more The low aqueous solubility of drug-like compounds has been described as one of the main problems causing the failure of new chemical entities in medicinal chemistry programmes. This paper describes our efforts to overcome lack of solubility and to produce a novel template protocol for forming salts of poorly soluble compounds. We prepared a series of 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one amine salts by using ultrasound irradiation. The poorly water-soluble molecule 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one was recently described as an inhibitor of the Mycobacterium tuberculosis orotate phosphoribosyltransferase enzyme. Salts of this molecule were prepared in 88–98 % yield from a mixture of 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one with primary and secondary amines (dimethylamine, diethylamine, dipropylamine, ethanolamine, 2-amino-2-methyl-1,3-propanediol, d-threoninol, pyrrolidine and piperidine) after ultrasound irradiation. The main advantages of this method are a significant reduction in reaction times and the use of a renewable solvent as a reaction medium. Thus the compounds are obtained after irradiation for 8 min in ethanol.Graphical abstract
Microbiology, 2009
Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tube... more Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The molecular mechanisms of infection and persistence have not been completely elucidated for this pathogen. Studies involving nucleoid-associated proteins (NAPs), which have been related to the control and influence of virulence genes in pathogenic bacteria, can help unveil the virulence process of M. tuberculosis. Here, we describe the initial characterization of an ORF for an M. tuberculosis putative NAP. The Rv3852 gene was cloned and expressed, and its product purified to homogeneity. A qualitative protein–DNA binding assay was carried out by gel-retardation and the protein affinity for specific DNA sequences was assessed quantitatively by surface plasmon resonance (SPR). A stoichiometry of 10 molecules of monomeric protein per molecule of DNA was determined. The monophasic apparent dissociation rate constant values increased to a saturable level as a function of protein c...
Journal of the Brazilian Chemical Society, 2010
A tuberculose (TB) continua sendo a principal causa de mortalidade devido a um único patógeno bac... more A tuberculose (TB) continua sendo a principal causa de mortalidade devido a um único patógeno bacteriano, o Mycobacterium tuberculosis. Há, portanto, a necessidade de desenvolvimento de novos agentes antimicobacterianos. A 2-trans-enoil-ACP(CoA) redutase (InhA) de M. tuberculosis é o principal alvo da isoniazida. Aqui nós apresentamos dados de equilíbrio e cinética em estado pré-estacionário da ligação do substrato 2-trans-dodecenoil-CoA à InhA. Os resultados demonstram cooperatividade homotrópica positiva da ligação deste substrato à InhA e um processo de associação bimolecular seguido por uma lenta isomerização do complexo binário enzima-substrato. Os dados aqui descritos devem auxiliar no desenho racional de novos inibidores de um alvo protéico validado e com potencial utilização no tratamento da TB. Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. There is a need for the development of new antimycobacterial agents. M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase (InhA) is the main target of isoniazid, the most prescribed anti-TB agent. Here we present pre-steady state kinetics and equilibrium data of 2-trans-dodecenoyl-CoA substrate binding to InhA. These results indicate both positive homotropic cooperativity upon substrate binding to InhA, and a bimolecular association process followed by a slow isomerization of the enzyme-substrate binary complex. The data here described should help the rational design of new agents against a validated and druggable protein target with potential anti-TB activity.
Journal of Structural Biology, 2006
In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatic... more In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 Ǻ resolution. The MtCS structure is similar to other CS structures, presenting b-a-b sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event.
Journal of Structural Biology, 2010
The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tub... more The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2 0-deoxycytidine for uridine and 2 0-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn 2+-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K m = 1004 lM and k cat = 4.8 s À1 for cytidine, and K m = 1059 lM and k cat = 3.5 s À1 for 2 0-deoxycytidine. The pH dependence of k cat and k cat /K M for cytidine indicate that protonation of a single ionizable group with apparent pK a value of 4.3 abolishes activity, and protonation of a group with pK a value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 Å resolution. Analysis of the crystallographic structure indicated the presence of a Zn 2+ coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.
Current Topics in Medicinal Chemistry, 2013
Cell Biochemistry and Biophysics, 2006
Crystallographic screening has been used to identify new inhibitors for potential target for drug... more Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.
BMC Research Notes, 2012
Background Tuberculosis (TB) still remains one of the most deadly infectious diseases in the worl... more Background Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli. Findings Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data. Conclusions The results suggest tha...
Biochemistry, 2006
ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsib... more ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of-ketoacyl-ACP to generate-hydroxyacyl-ACP and NADP +. In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C 4-proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse D 2 O V/K and an increase in D 2 O V when [4S-2 H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data.
Biochemistry, 2001
Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tub... more Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K i of 2.2 nM) followed by an overall dissociation constant (K i *) of 28 pM, yielding a K m /K i * value of 10 6. Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s-1 , while relaxation of the complex is slower at 1.4 × 10-3 s-1. The pH dependence of the K i value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K is of 0.39 µM) while the 1,4dideoxy-1,4-iminoribitol binds weakly (K is of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol () 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 µM) compared to 9-deazahypoxanthine alone, and by a factor of >10 8 compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [
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Papers by Diogenes santos silva Santos