RESUMO
5-Methylcytosine methyltransferases (m5C MTases) are known to be involved in the modification of RNA. Although these enzymes have been relatively well characterized in bacteria and eukarya, a complete understanding of the archaeal counterparts is lacking. In this study, the identification and characterization of archaeal RNA m5C MTases were performed. As a case study, a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, which possesses five putative RNA m5C MTases, was chosen. Among the five putative RNA m5C MTases, two proteins (PH0851 and PH1991) have been characterized as homologs of a bacterial rRNA MTase (RsmB) and eukaryal tRNA MTase (NSUN6), respectively. The in-depth characterization of the remaining three putative RNA m5C MTases (PH1078, PH1374, and PH1537) in this study suggests the presence of the signature architecture and catalytic residues plausibly involved in the binding of their cognate RNA substrates. Additionally, the results also suggest the existence of two RsmB-like proteins (PH0851 and PH1078) belonging to the same subfamily IV of m5C RNA MTase. However, the proteins PH1374 and PH1537 belong to the same subfamily V but bind to different substrates, rRNA and tRNA, respectively. The findings further indicate that archaeal RNA m5C MTases link those from bacteria and eukarya.Communicated by Ramaswamy H. Sarma.
RESUMO
The redox property of iron makes it an essential cofactor for numerous enzymes involved in various metabolic processes. In vertebrates, iron is attached to either heme molecules or with other circulatory proteins, making its accessibility restricted for bacterial pathogens residing inside the host. Due to this importance, there is always an ongoing battle between the host system and pathogens, known as nutritional immunity. To capture the bound iron from the human hosts, intracellular pathogens like Mycobacterium tuberculosis secrete siderophore molecules which are ultimately uptaken by versatile transport machinery such as ATP-binding cassette (ABC) transporters. Earlier reports have suggested the presence of a heme uptake protein MhuP (ORF id: Rv0265c) in M. tuberculosis, which transiently transfers the bound iron to the protein DppA for further heme transport by utilizing its cognate transport machinery (DppBCD). In the present study, we report the crystal structure of MhuP. The binding experiments of heme with MhuP suggest its specific nature. Molecular docking studies confirm the binding of the protein MhuP with heme as well as to the protein DppA. Thus, the results indicate the binding of heme to MhuP and its probable transient transport via the DppABCD transport system in M. tuberculosis.
Assuntos
Hemeproteínas , Mycobacterium tuberculosis , Tuberculose , Transportadores de Cassetes de Ligação de ATP/química , Animais , Proteínas de Bactérias/química , Heme/química , Hemeproteínas/metabolismo , Humanos , Ferro/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologiaRESUMO
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases known to degrade extracellular matrix (ECM). Being involved in many biological and physiological processes of tissue remodeling, MMPs play a crucial role in many pathological conditions such as arthritis, cancer, cardiovascular diseases, etc. Typically, MMPs possess a propeptide, a zinc-containing catalytic domain, a hinge region and a hemopexin domain. Based on their structural domain organization and substrates, MMPs are classified into six different classes, viz. collagenases, stromelysins, gelatinases, matrilysins, membrane-type and other MMPs. As per previous studies, a set of invariant water (IW) molecules of MMP-1 (a collagenase) play a significant role in stabilizing their catalytic domain. However, a functional role of IW molecule in other classes of MMPs has not been reported yet. Thus, in this study, IW molecules of MMPs from different classes were located and their plausible role(s) have been assigned. The results suggest that IW molecules anchor the structurally and functionally essential metal ions present in the vicinity of the active site of MMPs. Further, they (in)directly interlink different structural features and bridge the active site metal ions of MMPs. This study provides the key IW molecules that are structurally and functionally relevant to MMPs and hence, in turn, might facilitate the development of potent generalized inhibitor(s) against different classes of MMPs. Communicated by Ramaswamy H. Sarma.
Assuntos
Neoplasias , Água , Humanos , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Neoplasias/metabolismo , Mineração de Dados , Zinco/metabolismo , Matriz Extracelular/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologiaRESUMO
More than one third of proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria they are translocated by Na+/citrate symporters. In this study, the presence of a novel divalent-metal-ion-complexed citrate-uptake system that belongs to the primary active ABC transporter superfamily is reported. For uptake, the metal-ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains for their transport. This study reports crystal structures of an Mg2+-citrate-binding protein (MctA) from the Gram-negative thermophilic bacterium Thermus thermophilus HB8 in both apo and holo forms in the resolution range 1.63-2.50â Å. Despite binding various divalent metal ions, MctA possesses the coordination geometry to bind its physiological metal ion, Mg2+. The results also suggest an extended subclassification of cluster D SBPs, which are known to bind and transport divalent-metal-ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant MctA proteins suggests a gating mechanism of ligand entry following an `asymmetric domain movement' of the N-terminal domain for substrate binding.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Citratos/metabolismo , Magnésio/química , Proteínas/metabolismo , TermodinâmicaRESUMO
Mycobacterium tuberculosis, one of the major threats to mankind, requires micronutrients like metal ions for their survival and pathogenicity inside the host system. Intracellular pathogens such as M. tuberculosis have co-evolved to combat the nutritional immunity developed by the host. It has developed eminent mechanisms to sequester essential metal ions from the host system. One such prominent mechanism to scavenge metal ions to thrive in the host cell involves ATP-binding cassette (ABC) transporters, which transport metal ions (in free and/or complex forms) across the cell membrane. This study employs a high-throughput data mining analysis to identify open reading frames (ORFs) encoding metal uptake ABC transporters in M. tuberculosis H37Rv. In total, 19 ORFs resulting in seven ABC transport systems and two P-type ATPases were identified, which are potentially involved in the uptake of different metal ions. The results also suggest the existence of a subunit sharing mechanism in M. tuberculosis where the transmembrane and nucleotide binding domains are shared among different ABC transport systems indicating the import of multiple substrates via a single ABC transporter. Thus, this study reflects an overview of the repertoire of metal-specific ABC transport systems in M. tuberculosis H37Rv, providing potential therapeutic targets for the future.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais/metabolismo , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Domínio Catalítico , Mineração de Dados , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/genética , Fases de Leitura Aberta , Conformação ProteicaRESUMO
Organisms use a variety of carbohydrates and metabolic pathways in order to capitalize in their specific environments. Depending upon their habitat, organism employs different types of transporters to maintain the cellular nutritional balance via central metabolism. A major contributor in this process in bacteria is a carbohydrate ABC transporter. The focus of this study is to get an insight into the carbohydrate transport and metabolism of a hot-spring-dwelling bacterium Thermus thermophilus HB8. We applied high-throughput data-mining approaches for identification and characterization of carbohydrate ABC transporters in T. thermophilus HB8. This enabled the identification of 11 putative carbohydrate ABC transport systems. To identify the cognate ligands for these transporters, functional annotation was performed. However, scarcity of homologous-protein's function hinders the process of functional annotation. Thus, to overcome this limitation, we integrated the functional annotation of carbohydrate ABC transporters with their metabolic analysis. Our results demonstrate that out of 11 putative carbohydrate ABC transporters, six are involved in the sugar (four for monosaccharides and polysaccharides-degraded products and two for osmotic regulation), four in phospholipid precursor (namely UgpABCE) and the remaining one in purine uptake. Further, analysis suggests the existence of sharing mechanism of transmembrane domains (TMDs) and/or nucleotide-binding domains (NBDs) among the 11 carbohydrate ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metabolismo dos Carboidratos/fisiologia , Redes e Vias Metabólicas/fisiologia , Domínios Proteicos/fisiologia , Thermus thermophilus/fisiologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos/genética , Simulação de Acoplamento Molecular , Fosfolipídeos/metabolismo , Ligação Proteica , Purinas/metabolismo , Especificidade por SubstratoRESUMO
Micronutrients such as metal ions are indispensable for the growth and survival of microorganisms in assorted environmental niches. However, change in cellular concentration of metal ions is pernicious for an organism; thus metal ion homeostasis is crucial for their survival and growth. An eminent mechanism for maintaining metal ion homeostasis in microorganisms is ATP-binding cassette (ABC) transporters, which transport metal ions in their ionic/complex forms across the cell membrane. For the uptake, metals are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains (TMDs) for their transport. In this work, a high-throughput data mining analysis has been performed to identify open reading frames (ORFs) encoding metal-specific ABC transporters in a thermophilic bacterium Thermus thermophilus HB8. In total, 22 ORFs resulting in eight ABC transport systems were identified, which are potentially involved in the uptake of metal ions. This study suggests that three out of eight metal-specific ABC import systems are specific to iron ions. Among the remaining five, two are particular to divalent metal ions such as Mg2+ and Zn2+/Mn2+, another two are for tetrahedral oxyanions such as MoO42- and WO42- and the remaining one imports cyanocobalamin (vitamin B12). Besides these, the results of this study demonstrate the existence of a mechanism where TMD and NBD components are shared among different ABC transport systems hinting that multiple substrates can be imported via a single transporter. This study thus provides the first ever preliminary glimpse into the entire repertoire of metal uptake ABC transporters in a thermophilic organism.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias , Metais Pesados , Thermus thermophilus , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ligantes , Metais Pesados/química , Metais Pesados/metabolismo , Micronutrientes , Simulação de Acoplamento Molecular , Filogenia , Ligação Proteica , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMO
BACKGROUND: Aspergillus fumigatus R1 produced xylanase under submerged fermentation which degrades the complex hemicelluloses contained in agricultural substrates. Xylanases have gained considerable attention because of their tremendous applications in industries. The purpose of our study was to purify xylanase and study its biochemical properties. We have predicted the secondary structure of purified xylanase and evaluated its active site residues and substrate binding sites based on the global and local structural similarity. RESULTS: Various microorganisms were isolated from Puducherry soil and screened by Congo-red test. The best isolate was identified to be Aspergillus fumigatus R1. The production kinetics showed the highest xylanase production (208 IU/ml) by this organism in 96 h using 1 % rice bran as the only carbon source. The purification of extracellular xylanase was carried out by fractional ammonium sulphate precipitation (30-55 %), followed by extensive dialysis and Bio-Gel P-60 Gel-filtration chromatography. The enzyme was purified 58.10 folds with a specific activity of 38196.22 IU/mg. The biochemical characterization of the pure enzyme was carried out for its optimum pH and temperature (5.0 and 50(0)C), pH and temperature stability, molecular mass (Mr) (24.5 kDa) and pI (6.29). The complete sequence of protein was obtained by mass spectrometry analysis. Apparent Km and Vmax values of the xylanase for birchwood xylan were 11.66 mg/ml and 87.6 µmol min(-1) mg(-1) respectively. CONCLUSION: Purified xylanase was analyzed by mass-spectrometry which revealed 2 unique peptides. Xylanase under current study showed significant production using agricultural residues and a broad range of pH stability in the alkaline region. Xylanase produced by Aspergillus fumigatus R1 could serve as the enzyme of choice in industries.
Assuntos
Aspergillus fumigatus/enzimologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Microbiologia do Solo , TemperaturaRESUMO
About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A2, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C2B domain) bound to seven different divalent metal ions (Ca(2+), Mg(2+), Zn(2+), Fe(2+), Cu(2+), Ba(2+), and Sr(2+), respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe(2+) and Cu(2+), respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.