Evolutionary trends indicate a coherent organization of sap operons.

Human hosts possess a complex network of immune responses against microbial pathogens. The production of antimicrobial peptides (AMPs), which target the pathogen cell membranes and inhibit them from inhabiting the hosts, is one such mechanism. However, pathogens have evolved systems that encounter these host-produced AMPs. The Sap (sensitivity to antimicrobial peptides) transporter uptakes AMPs inside the microbial cell and proteolytically degrades them. The Sap transporters comprise five subunits encoded by genes in an operon. Despite its ubiquitous nature, its subunits are not found to be in tandem with many organisms. In this study, a total of 421 Sap transporters were analyzed for their operonic arrangement. Out of 421, a total of 352 operons were found to be in consensus arrangement, while the remaining 69 show a varying arrangement of genes. The analysis of the intergenic distance between the subunits of the sap operon suggests a signature pattern with sapAB (-4), sapBC (-14), sapCD (-1), and sapDF (-4 to 1). An evolutionary analysis of these operons favors the consensus arrangement of the Sap transporter systems, substantiating its prevalence in most of the Gram-negative pathogens. Overall, this study provides insight into bacterial evolution, favoring the maintenance of the genetic organization of essential pathogenicity factors.

Distinct characteristics of putative archaeal 5-methylcytosine RNA methyltransferases unveil their substrate specificities and evolutionary ancestries.

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.

The Structural Features of MlaD Illuminate its Unique Ligand-Transporting Mechanism and Ancestry.

The membrane-associated solute-binding protein (SBP) MlaD of the maintenance of lipid asymmetry (Mla) system has been reported to help the transport of phospholipids (PLs) between the outer and inner membranes of Gram-negative bacteria. Despite the availability of structural information, the molecular mechanism underlying the transport of PLs and the ancestry of the protein MlaD remain unclear. In this study, we report the crystal structures of the periplasmic region of MlaD from Escherichia coli (EcMlaD) at a resolution range of 2.3-3.2 Å. The EcMlaD protomer consists of two distinct regions, viz. N-terminal ß-barrel fold consisting of seven strands (referred to as MlaD domain) and C-terminal α-helical domain (HD). The protein EcMlaD oligomerizes to give rise to a homo-hexameric ring with a central channel that is hydrophobic and continuous with a variable diameter. Interestingly, the structural analysis revealed that the HD, instead of the MlaD domain, plays a critical role in determining the oligomeric state of the protein. Based on the analysis of available structural information, we propose a working mechanism of PL transport, viz. "asymmetric protomer movement (APM)". Wherein half of the EcMlaD hexamer would rise in the periplasmic side along with an outward movement of pore loops, resulting in the change of the central channel geometry. Furthermore, this study highlights that, unlike typical SBPs, EcMlaD possesses a fold similar to EF/AMT-type beta(6)-barrel and a unique ancestry. Altogether, the findings firmly establish EcMlaD to be a non-canonical SBP with a unique ligand-transport mechanism.

Dual-target drugs against Leishmania donovani for potential novel therapeutics.

Antioxidant defense mechanisms are important for a parasite to overcome oxidative stress and survive within host macrophage cells. Mitochondrial iron superoxide dismutase A (FeSODA) and trypanothione reductase (TR) are critical enzymes in the antioxidant defense mechanism of Leishmania donovani. FeSODA is responsible for neutralizing reactive oxygen species in mitochondria, while TR is responsible for reducing trypanothione, the molecules that help the parasite fight oxidative stress in Leishmania. In this study, we used multitarget ligands to inhibit both the FeSODA and TR enzymes. We combined structure-based drug design using virtual screening approach to find inhibitors against both the targets. The ZINC15 database of biogenic compounds was utilized to extract drugs-like molecules against leishmaniasis. The compounds were screened by standard precision (SP) and extra precision (XP) docking methods. Two compounds, ZINC000008876351 and ZINC000253403245, were selected based on molecular docking based on the binding affinity for both the targets. The screened molecules ZINC000008876351 and ZINC000253403245 showed strong hydrogen bonding with the target proteins according to the Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) techniques. These two compounds were also experimentally investigated on promastigotes stage of L. donovani. Under in vitro condition, the compounds show inhibitory effects on L. donovani promastigotes with IC50 values of 24.82 ± 0.61 µM for ZINC000008876351 and 7.52 ± 0.17 µM for ZINC000253403245. Thus, the screened compounds seem to have good potential as therapeutic candidates for leishmaniasis.

Operon Finder: A Deep Learning-based Web Server for Accurate Prediction of Prokaryotic Operons.

Operons are groups of consecutive genes that transcribe together under the regulation of a common promoter. They influence protein regulation and various physiological pathways, making their accurate detection desirable. The detection of operons through experimental means is a laborious and financially intensive process. Therefore, human experts predict potential operons utilizing their prior knowledge of the genetic organization and functional correlation of the genes. However, with the rise in the number of completely sequenced genomes, the development of automated algorithms, tools, and web servers is highly preferred over manual detection for operon prediction. Currently available state-of-the-art algorithms use a deep learning-based model to predict if the adjacent genes belong to the same operons in the given genome. However, these require an understanding of programming knowledge and computational skills, making them not-very user friendly. In this study, we developed a user-friendly web service, Operon Finder, for on-the-fly prediction of operons using the deep learning method. The interface provides a facility for genome search, operon live-filtering, viewing operonic DNA sequences, downloading predicted results, and links for data retrieval from the NCBI (National Center for Biotechnology Information) database. The web server is available at https://www.iitg.ac.in/spkanaujia/operonfinder.html. The experimental methods and the implementation details are publicly available at https://github.com/SPKlab/Operon-Finder.

Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component.

The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1-71 amino acids) and C-terminal (72-90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.

Role of an orphan substrate-binding protein MhuP in transient heme transfer in Mycobacterium tuberculosis.

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.

Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation.

Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma.

Structural and functional role of invariant water molecules in matrix metalloproteinases: a data-mining approach.

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.

Structural and thermodynamic insights into a novel Mg2+-citrate-binding protein from the ABC transporter superfamily.

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 1.63-2.50 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.

An updated classification and mechanistic insights into ligand binding of the substrate-binding proteins.

Substrate-binding proteins (SBPs) mediate ligand translocation and have been classified into seven clusters (A-G). Although the substrate specificities of these clusters are known to some extent, their ligand-binding mechanism(s) remain(s) incompletely understood. In this study, the list of SBPs belonging to different clusters was updated (764 SBPs) compared to the previously reported study (504 SBPs). Furthermore, a new cluster referred to as cluster H was identified. Results reveal that SBPs follow different ligand-binding mechanisms. Intriguingly, the majority of the SBPs follow the 'one domain movement' rather than the well-known 'Venus Fly-trap' mechanism. Moreover, SBPs of a few clusters display subdomain conformational movement rather than the complete movement of the N- and C-terminal domains.

Conserved features of the MlaD domain aid the trafficking of hydrophobic molecules.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids between the inner (IM) and outer membrane. The Mla system utilizes a unique IM-associated periplasmic solute-binding protein, MlaD, which possesses a conserved domain, MlaD domain. While proteins carrying the MlaD domain are known to be primarily involved in the trafficking of hydrophobic molecules, not much is known about this domain itself. Thus, in this study, the characterization of the MlaD domain employing bioinformatics analysis is reported. The profiling of the MlaD domain of different architectures reveals the abundance of glycine and hydrophobic residues and the lack of cysteine residues. The domain possesses a conserved N-terminal region and a well-preserved glycine residue that constitutes a consensus motif across different architectures. Phylogenetic analysis shows that the MlaD domain archetypes are evolutionarily closer and marked by the conservation of a functionally crucial pore loop located at the C-terminal region. The study also establishes the critical role of the domain-associated permeases and the driving forces governing the transport of hydrophobic molecules. This sheds sufficient light on the structure-function-evolutionary relationship of MlaD domain. The hexameric interface analysis reveals that the MlaD domain itself is not a sole player in the oligomerization of the proteins. Further, an operonic and interactome map analysis reveals that the Mla and the Mce systems are dependent on the structural homologs of the nuclear transport factor 2 superfamily.

Identification and characterization of metal uptake ABC transporters in Mycobacterium tuberculosis unveil their ligand specificity.

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.

Structural and thermodynamic insights into the novel dinucleotide-binding protein of ABC transporter unveils its moonlighting function.

Substrate (or solute)-binding proteins (SBPs) selectively bind the target ligands and deliver them to the ATP-binding cassette (ABC) transport system for their translocation. Irrespective of the different types of ligands, SBPs are structurally conserved. A wealth of structural details of SBPs bound to different types of ligands and the physiological basis of their import are available; however, the uptake mechanism of nucleotides is still deficient. In this study, we elucidated the structural details of an SBP endogenously bound to a novel ligand, a derivative of uridylyl-3'-5'-phospho-guanosine (U3G); thus, we named it a U3G-binding protein (U3GBP). To the best of our knowledge, this is the first report of U3G (and a dinucleotide) binding to the SBP of ABC transport system, and thus, U3GBP is classified as a first member of subcluster D-I SBPs. Thermodynamic data also suggest that U3GBP can bind phospholipid precursor sn-glycerophosphocholine (GPC) at a site other than the active site. Moreover, a combination of mutagenic and structural information reveals that the protein U3GBP follows the well-known 'Venus Fly-trap' mechanism for dinucleotide binding. DATABASES: Structural data are available in RCSB Protein Data Bank under the accession number(s) 7C0F, 7C0K, 7C0L, 7C0O, 7C0R, 7C0S, 7C0T, 7C0U, 7C0V, 7C0W, 7C0X, 7C0Y, 7C0Z, 7C14, 7C15, 7C16, 7C19, and 7C1B.

Acyldepsipeptide activated ClpP1P2 macromolecule of Leptospira, an ideal Achilles' heel to hamper the cell survival and deregulate ClpP proteolytic activity.

Antibiotic acyldepsipeptide (ADEP) targets the bacterial ClpP serine protease and can inhibit the growth of numerous bacterial species by activating/dysregulating the protease activity within the cell. The spirochete Leptospira interrogans harbors two ClpP isoforms (LepClpP1 and LepClpP2). Supplementation of ADEP in the Leptospira growth medium resulted in the inhibition of bacterial growth. The ADEP mediated activation of the LepClpP mixture was dependent on the time allowed for the self-assembly of LepClpP1 and LepClpP2. The dynamic light scattering of the LepClpP mixture in the presence of the ADEP indicated a conformational transformation of the LepClpP machinery. Serine 98, a catalytic triad residue of the LepClpP1 in the LepClpP1P2 heterocomplex, was critical for the ADEP mediated activation. The computational prototype of the LepClpP1P2 structure suggested that the hydrophobic pockets wherein the ADEPs or the physiological chaperone ClpX predominantly dock are exclusively present in the LepClpP2 heptamer. Using the ADEP as a tool, this investigation provides an insight into the molecular function of the LepClpP1P2 in a coalition with its ATPase chaperone LepClpX. The shreds of the evidence illustrated in this investigation verify that ADEP1 possesses the ability to control the LepClpP system in an unconventional approach than the other organisms.

Exploiting the rationale behind substrate recognition by promiscuous thermophilic NDP-sugar pyrophosphorylase for expanding glycorandomization: an in silico study.

The fundamental substrates for protein glycosylation are provided by a group of enzymes known as NDP-sugar pyrophosphorylases (NSPases) which utilize nucleotide triphosphate (NTP) and sugar 1-phosphate to catalyze the formation of nucleotide diphospho-sugar (NDP-sugar). The promiscuous nature of NSPases is often exploited during chemoenzymatic glycorandomization in the pursuit of novel therapeutics. However, till date, the number of inherently promiscuous NSPases reported and the rationale behind their promiscuity is meager. In this study, we have identified a set of NSPases from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 to identify probable candidates for glycorandomization. We identified a set of NSPases that include both substrate-specific and substrate-promiscuous NSPases with a visible predominance of the latter group. The rationale behind the promiscuity (or specificity) vividly lies in the repertoire of amino acid residues that assemble the active site for recognition of the substrate moiety. Furthermore, the absence of a function-specific auxiliary domain promotes substrate promiscuity in NSPases. This study, thus, provides a novel set of thermophilic NSPases that can be employed for chemoenzymatic glycorandomization. More importantly, identification of the residues that render substrate promiscuity (or specificity) would assist in sequence-based rational engineering of NSPases for enhanced glycorandomization. Communicated by Ramaswamy H. Sarma.

Conformational Trapping of a ß-Glucosides-Binding Protein Unveils the Selective Two-Step Ligand-Binding Mechanism of ABC Importers.

Substrate-binding proteins (SBPs), selectively capture ligand(s) and ensure their translocation via its cognate ATP-binding cassette (ABC) import system. SBPs bind their cognate ligand(s) via an induced-fit mechanism known as the "Venus Fly-trap"; however, this mechanism lacks the atomic details of all conformational landscape as the confirmatory evidence(s) in its support. In this study, we delineate the atomic details of an SBP, ß-glucosides-binding protein (ßGlyBP) from Thermus thermophilus HB8. The protein ßGlyBP is multi-specific and binds to different types of ß-glucosides varying in their glycosidic linkages viz. ß-1,2; ß-1,3; ß-1,4 and ß-1,6 with a degree of polymerization of 2-5 glucosyl units. Structurally, the protein ßGlyBP possesses four subdomains (N1, N2, C1 and C2). The unliganded protein ßGlyBP remains in an open state, which closes upon binding to sophorose (SOP2), laminari-oligosaccharides (LAMn), cello-oligosaccharides (CELn), and gentiobiose (GEN2). This study reports, for the first time, four different structural states (open-unliganded, partial-open-unliganded, open-liganded and closed-liganded) of the protein ßGlyBP, revealing its conformational changes upon ligand binding and suggesting a two-step induced-fit mechanism. Further, results suggest that the conformational changes of N1 and C1 subdomains drive the ligand binding, unlike that of the whole N- and C-terminal domains (NTD and CTD) as known in the "Venus Fly-trap" mechanism. Additionally, profiling of stereo-selection mechanism for α- and ß-glucosides reveals that in the ligand-binding site four secondary structural elements (L1, H1, H2 and H3) drive the ligand selection. In summary, results demonstrate that the details of conformational changes and ligand selection are pre-encoded in the SBPs.

Structural and thermodynamic correlation illuminates the selective transport mechanism of disaccharide α-glycosides through ABC transporter.

Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an α-glycoside-binding protein (αGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide α-glycosides namely trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6) and glucose within a resolution range of 1.6-2.0 Å. Despite transporting multiple types of sugars, αGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide α-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide α-glycosides) from the active site of the protein. Comparative assessment of open and closed conformations of αGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of αGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession number(s) 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE.

Role of Structural Features in Oligomerization, Active-Site Integrity and Ligand Binding of Ribose-1,5-Bisphosphate Isomerase.

Pentose bisphosphate pathway, exclusively found in archaea, is similar to the pentose phosphate pathway present in bacteria and eukarya. In pentose bisphosphate pathway, the conversion of ribose moieties of nucleosides into 3-phosphoglycerate (3-PGA) involves multiple steps; one of them being the conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) catalyzed by an enzyme ribose-1,5-bisphosphate isomerase (R15Pi). The availability of the three-dimensional structure of R15Pi had facilitated the understanding of various structural and functional aspects of the enzyme. Nevertheless, the structure of R15Pi also left several significant questions unanswered that would aid in understanding the structure-function relationship of the enzyme. Thus, we have taken up a computational approach to further understand the role of various structural features of the enzyme R15Pi. Results obtained from molecular dynamics (MD) simulations aided in understanding the obligation of the enzyme R15Pi to oligomerize and also in deciphering the role of catalytic residue(s) in structural stability. Identification of invariant water molecules of the enzyme R15Pi helped in discerning their significance at the active-site pocket and structurally important regions. Further, molecular docking studies allowed the identification of the amino acid residues essential for holding the substrate (R15P) or product (RuBP) in the vicinity of the active site of the enzyme R15Pi. Interestingly, results of the molecular docking studies assisted in the identification of an "alternate binding site" for the substrate, R15P. Finally, based on these results, we propose a mechanism of "substrate sliding" for the enzyme R15Pi.

Identification and characterization of ABC transporters for carbohydrate uptake in Thermus thermophilus HB8.

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.