TY - JOUR T1 - A dimer between monomers and hexamers-Oligomeric variations in glucosamine-6-phosphate deaminase family. JF - PLoS One Y1 - 2023 A1 - Srinivasachari, Sathya A1 - Tiwari, Vikas R A1 - Kharbanda, Tripti A1 - Sowdamini, Ramanathan A1 - Subramanian, Ramaswamy KW - Aldose-Ketose Isomerases KW - Bacterial Proteins KW - Haemophilus influenzae KW - N-Acetylneuraminic Acid KW - Pasteurella multocida KW - Polymers AB -

In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. Both Hi-NagB and Pm-NagB contain the C-terminal helix implicated as essential for hexamer formation. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. The conservation of the dimer interface suggests that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.

VL - 18 IS - 1 ER - TY - JOUR T1 - Modulation of biliverdin dynamics and spectral properties by Sandercyanin. JF - RSC Adv Y1 - 2022 A1 - Ghosh, Swagatha A1 - Mondal, Sayan A1 - Yadav, Keerti A1 - Aggarwal, Shantanu A1 - Schaefer, Wayne F A1 - Narayana, Chandrabhas A1 - Subramanian, Ramaswamy AB -

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

VL - 12 IS - 31 ER - TY - JOUR T1 - Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea. JF - Glycobiology Y1 - 2021 A1 - Subramanian, Sabarinath Peruvemba A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Subramanian, Ramaswamy AB -

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix, and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and di-substituted O-GalNAc structures. Extensions made of hexoses and 3-O methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, sixty genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. While a majority of the genes involved in mucin-type O-glycan biosynthesis was highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

ER - TY - JOUR T1 - A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase. JF - Angew Chem Int Ed Engl Y1 - 2020 A1 - Arya, Chetan Kumar A1 - Yadav, Swati A1 - Fine, Jonathan A1 - Casanal, Ana A1 - Chopra, Gaurav A1 - Ramanathan, Gurunath A1 - Vinothkumar, Kutti R A1 - Subramanian, Ramaswamy AB -

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

VL - 59 IS - 39 ER - TY - JOUR T1 - Comparison of CryoEM and X-ray structures of dimethylformamidase. JF - Prog Biophys Mol Biol Y1 - 2020 A1 - Vinothkumar, Kutti R A1 - Arya, Chetan Kumar A1 - Ramanathan, Gurunath A1 - Subramanian, Ramaswamy AB -

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 °C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

ER - TY - JOUR T1 - A perspective on challenges and opportunities in characterizing oral cancer stem cells. JF - Front Biosci (Landmark Ed) Y1 - 2020 A1 - Sadasivam, Subhashini A1 - Subramanian, Ramaswamy AB -

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) represent a minority population of cells in a tumor that can self-renew and re-create the heterogeneity of the entire tumor. Cell lines, patient-derived tumor cells, and patient-derived xenografts have all been used to isolate presumptive CSC populations from different tumor types. Because of their purported roles in tumor recurrence and prognosis, numerous efforts have centered around reliably identifying CSCs using cell surface markers, and in using genomics tools to identify molecular features unique to these cells. In this brief review, we will discuss different markers, CD44, ALDH1, CD271 and others that have used for identifying and isolating CSCs from primary head & neck and oral squamous cell carcinomas. In particular, we focus on the challenges associated with these experiments as this will be useful to researchers attempting similar isolations. We also discuss some important molecular features gleaned from studying these CSCs such as the expression of stem cell-related markers, loss of cell adhesion and terminal differentiation markers, and the presence of both epithelial and epithelial-to-mesenchymal transition (EMT) features.

VL - 25 ER - TY - JOUR T1 - The resolution revolution reaches India. JF - Biophys Rev Y1 - 2019 A1 - Subramanian, Ramaswamy A1 - Mayor, Satyajit A1 - Vinothkumar, Kutti R ER - TY - JOUR T1 - Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae. JF - Acta Crystallogr D Struct Biol Y1 - 2019 A1 - Bose, Sucharita A1 - Purkait, Debayan A1 - Joseph, Deepthi A1 - Nayak, Vinod A1 - Subramanian, Ramaswamy KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Catalytic Domain KW - Crystallization KW - Crystallography, X-Ray KW - Cytidine Diphosphate KW - Cytidine Monophosphate N-Acetylneuraminic Acid KW - Cytidine Triphosphate KW - N-Acylneuraminate Cytidylyltransferase KW - Protein Interaction Domains and Motifs KW - Protein Structure, Quaternary KW - Sialic Acids KW - Vibrio cholerae AB -

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

VL - 75 IS - Pt 6 ER - TY - JOUR T1 - Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose -glycans in the planarian . JF - J Biol Chem Y1 - 2018 A1 - Subramanian, Sabarinath Peruvemba A1 - Babu, Ponnusamy A1 - Palakodeti, Dasaradhi A1 - Subramanian, Ramaswamy AB -

Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type -glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of -linked oligosaccharides from the planarian revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. -Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create >60 different glycoforms in These results contribute greatly to our understanding of -glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in

VL - 293 IS - 18 ER -