Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits.

5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.

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.

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.

In silico analysis of 5'-UTRs highlights the prevalence of Shine-Dalgarno and leaderless-dependent mechanisms of translation initiation in bacteria and archaea, respectively.

In prokaryotes, a heterogeneous set of protein translation initiation mechanisms such as Shine-Dalgarno (SD) sequence-dependent, SD sequence-independent or ribosomal protein S1 mediated and leaderless transcript-dependent exists. To estimate the distribution of coding sequences employing a particular translation initiation mechanism, a total of 107 prokaryotic genomes were analysed using in silico approaches. Analysis of 5'-untranslated regions (UTRs) of genes reveals the existence of three types of mRNAs described as transcripts with and without SD motif and leaderless transcripts. Our results indicate that although all the three types of translation initiation mechanisms are widespread among prokaryotes, the number of SD-dependent genes in bacteria is higher than that of archaea. In contrast, archaea contain a significantly higher number of leaderless genes than SD-led genes. The correlation analysis between genome size and SD-led & leaderless genes suggests that the SD-led genes are decreasing (increasing) with genome size in bacteria (archaea). However, the leaderless genes are increasing (decreasing) in bacteria (archaea) with genome size. Moreover, an analysis of the start-codon biasness confirms that among ATG, GTG and TTG codons, ATG is indeed the most preferred codon at the translation initiation site in most of the coding sequences. In leaderless genes, however, the codons GTG and TTG are also observed at the translation initiation site in some species contradicting earlier studies which suggested the usage of only ATG codon. Henceforth, the conventional mechanism of translation initiation cannot be generalized as an exclusive way of initiating the process of protein biosynthesis in prokaryotes.

Structural and functional characterization of archaeal DIMT1 unveils distinct protein dynamics essential for efficient catalysis.

Dimethyladenosine transferase 1 (DIMT1), an ortholog of bacterial KsgA is a conserved protein that assists in ribosome biogenesis by modifying two successive adenosine bases near the 3' end of small subunit (SSU) rRNA. Although KsgA/DIMT1 proteins have been characterized in bacteria and eukaryotes, they are yet unexplored in archaea. Also, their dynamics are not well understood. Here, we structurally and functionally characterized the apo and holo forms of archaeal DIMT1 from Pyrococcus horikoshii. Wild-type protein and mutants were analyzed to capture different transition states, including open, closed, and intermediate states. This study reports a unique inter-domain movement that is needed for substrate (RNA) positioning in the catalytic pocket, and is only observed in the presence of the cognate cofactors S-adenosyl-L-methionine (SAM) or S-adenosyl-L-homocysteine (SAH). The binding of the inhibitor sinefungine, an analog of SAM or SAH, to archaeal DIMT1 blocks the catalytic pocket and renders the enzyme inactive.

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.

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.

Development of novel dual-target drugs against visceral leishmaniasis and combinational study with miltefosine.

The dual-target inhibitors (ZINC000008876351 and ZINC000253403245) were identified by utilizing an advanced computational drug discovery method by targeting two critical enzymes such as FeSODA (Iron superoxide dismutase) and TryR (Trypanothione reductase) within the antioxidant defense system of Leishmania donovani (Ld). In vitro enzyme inhibition kinetics reveals that both the compound's ability to inhibit the function of enzyme LdFeSODA and LdTryR with inhibition constant (Ki) value in the low µM range. Flow cytometry analysis, specifically at IC50 and 2X IC50 doses of both the compounds, the intracellular ROS was significantly increased as compared to the untreated control. The compounds ZINC000253403245 and ZINC000008876351 exhibited strong anti-leishmanial activity in a dose-dependent manner against both the promastigote and amastigote stages of the parasite. The data indicate that these molecules hold promise as potential anti-leishmanial agents for developing new treatments against visceral leishmaniasis, specifically targeting the LdFeSODA and LdTryR enzymes. Additionally, the in vitro MTT assay shows that combining these compounds with miltefosine produces a synergistic effect compared to miltefosine alone. This suggests that the compounds can boost miltefosine's effectiveness by synergistically inhibiting the growth of L. donovani promastigotes. Given the emergence of miltefosine resistance in some Leishmania strains, these findings are particularly significant.

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.

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.

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 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.

Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms.

Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Šresolution, space group P2(1)) and Aquifex aeolicus (AaMogA; 1.70 Šresolution, space group P2(1) and 1.90 Šresolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein-protein complex of MogA and MoeA has been proposed and the residues involved in protein-protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.

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.

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.

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.

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.