RESUMO
WHO reported 10.4 million new tuberculosis (TB) cases and 1.8 million deaths in 2015, making M. tuberculosis the most successful human pathogen with highest mortality among infectious diseases [1,2]. Drug-resistant TB is a major threat to global TB control [2,3]. Recently Torres et al. [4] identified 14 novel substitutions in M. tuberculosis-KatG (the enzyme associated with resistance to isoniazid-an important first-line anti-TB drug) and demonstrated that 12 of the 14 can cause INH-resistance in M. smegmatis. This study presents an in silico structure-based analysis of these 14 amino acid substitutions using homology models and x-ray crystal structures (when available) in M. tuberculosis. Our models demonstrate that several of these mutations cluster around three openings in the KatG tertiary structure which appear to initiate channels to the heme group at the catalytic center of the enzyme. We studied the effects of these mutations on the tertiary structure of KatG, focusing on conformational changes in the three channels in the protein structure. Our results suggest that the 14 novel mutations sufficiently restrict one or more of these access channels, thus potentially preventing INH from reaching the catalytic heme. These observations provide valuable insights into the structure-based origins of INH resistance and provide testable hypotheses for future experimental studies.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catalase/química , Catalase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Genótipo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Fenótipo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Dihydrofolate reductase (DHFR), a key enzyme in tetrahydrofolate-mediated biosynthetic pathways, has a structural motif known to be highly conserved over a wide range of organisms. Given its critical role in purine and amino acid synthesis, DHFR is a well established therapeutic target for treating a wide range of prokaryotic and eukaryotic infections as well as certain types of cancer. Here we present a structural-based computer analysis of bacterial (Bacilli) and plasmid DHFR evolution. We generated a structure-based sequence alignment using 7 wild-type DHFR x-ray crystal structures obtained from the RCSB Protein Data Bank and 350 chromosomal and plasmid homology models we generated from sequences obtained from the NCBI Protein Database. We used these alignments to compare active site and non-active site conservation in terms of amino acid residues, secondary structure and amino acid residue class. With respect to amino acid sequences and residue classes, active-site positions in both plasmid and chromosomal DHFR are significantly more conserved than non-active site positions. Secondary structure conservation was similar for active site and non-active site positions. Plasmid-encoded DHFR proteins have greater degree of sequence and residue class conservation, particularly in sequence positions associated with a network of concerted protein motions, than chromosomal-encoded DHFR proteins. These structure-based were used to build DHFR specific phylogenetic trees from which evidence for horizontal gene transfer was identified.
Assuntos
Evolução Molecular , Conformação Proteica , Homologia Estrutural de Proteína , Tetra-Hidrofolato Desidrogenase/química , Sequência de Aminoácidos/genética , Bacillus/química , Bacillus/genética , Sítios de Ligação , Domínio Catalítico/genética , Cristalografia por Raios X , Modelos Moleculares , Filogenia , Plasmídeos/química , Plasmídeos/genética , Estrutura Secundária de Proteína , Alinhamento de Sequência , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The three-dimensional structure of acetylated virginiamycin M(1) (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90 degrees from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2' hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found; the loss of this binding interaction partly accounts for the release of the product from the active site.
Assuntos
Solventes/química , Virginiamicina/química , Acetilação , Sítios de Ligação , Modelos Moleculares , Conformação MolecularRESUMO
Systems biology, metabolomics, metabolic engineering, and other recent developments in biochemistry suggest that future biochemists will require a detailed familiarity with the compounds and pathways of intermediary metabolism and their biochemical control. The challenge to the biochemistry instructor is the presentation of metabolic pathways in a manner that allows student creativity in learning the pathways and their components. One approach that does permit the use of problem solving for the study of metabolic pathways involves following the fate of (13) C, (14) C, or (15) N labels, presented originally in the structure of an important metabolic starting compound, through relevant metabolic pathways. This method allows the presentation and study of problems in which such an isotopic label can be traced through two or more metabolic pathways, thus illustrating how these pathways are interconnected. The understanding that all the pathways of intermediary metabolism are interconnected provides opportunities to discuss their metabolic control by such mechanisms as signaling, feedback inhibition, location in organelles, coenzyme levels, and coenzyme recycling rates. The method is illustrated by following the fate of (14) C labels through anaerobic glycolysis, gluconeogenesis, and fatty acid transport, ß-oxidation, and ketone body formation. Cholesterol biosynthesis and heme formation are used to show that presentations of long and complex pathways can demonstrate important biochemical concepts by following the fate of an isotopic label using only the most important intermediates. Problems based on tracing radioactive labels through one or more metabolic pathways allow the use of cooperative learning techniques.
RESUMO
Human mammary carcinoma MCF-7 cell line responsiveness to the pteridines xanthopterin and isoxanthopterin was studied using the MTS assay for measurement of cell viability. The pteridines were tested at concentrations ranging from 7.8 to 500 microM singly and in 11 isoxanthopterin:xanthopterin ratios. IC50s of xanthopterin and isoxanthopterin were 109+/-13 microM (mean+/-SEM of y estimate) and 103+/-9 microM, respectively. The IC50 values for pteridine mixtures were similar although 3:1 and 4:1 isoxanthopterin:xanthopterin ratios seemed slightly more cytotoxic than other mixtures. However, ANOVA revealed no statistical differences in the cytotoxicity of mixtures.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Xantopterina/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Concentração Inibidora 50 , Xantopterina/toxicidadeRESUMO
The antibiotic virginiamycin is a combination of two molecules, virginiamycin M(1) (VM1) and virginiamycin S(1) (VS1) or analogues, which function synergistically by binding to bacterial ribosomes and inhibiting bacterial protein synthesis. Both VM1 and VS1 dissolve poorly in water and are soluble in more hydrophobic solvents. We have recently reported that the 3D conformation of VM1 in CDCl(3) solution differs markedly from the conformation bound to a VM1 binding enzyme and to 50S ribosomes as found by X-ray crystallographic studies. We now report the results of further NMR studies and subsequent molecular modeling of VM1 dissolved in CD(3)CN/H(2)O and compare the structure with that in CD(3)OD and CDCl(3). The conformations of VM1 in CD(3)CN/H(2)O, CD(3)OD and CDCl(3) differ substantially from one another and from the bound form, with the aqueous form most like the bound structure. We propose that the flexibility of the VM1 molecule in response to environmental conditions contributes to its effectiveness as an antibiotic.
Assuntos
Antibacterianos/química , Biofísica/métodos , Estreptogramina A/química , Acetiltransferases/metabolismo , Sítios de Ligação , Calibragem , Cristalografia por Raios X , Meio Ambiente , Substâncias Macromoleculares/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Prolina/química , Conformação Proteica , Ribossomos/química , Software , Solventes , Streptomyces/metabolismo , TemperaturaRESUMO
The streptogramins are antibiotics which act by binding two different components at separate nearby sites on the bacterial 50S ribosome, inhibiting protein synthesis. The first component, a macrolactone, is common to many of the streptogramin antibiotics and, thus, is referred to by many names including virginiamycin M1(VM1), pristinamycin IIA, ostreogrycin A and streptogramin A. X-Ray crystallographic studies of VM1 bound to ribosomes and to a deactivating enzyme show a different conformation to that of VM1 in chloroform solution. We now report the results of high resolution 2D NMR experiments that show that the conformation of VM1 in dimethyl sulfoxide and methanol differs from both that in chloroform solution and in the bound form. The 3D structure and the 1H NMR and 13C NMR chemical shifts of VM1 in dimethyl sulfoxide and methanol are described.