Velavan Ramasamy - Academia.edu (original) (raw)

Papers by Velavan Ramasamy

Research paper thumbnail of with a Pentasacc n

T1, the Km of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, an... more T1, the Km of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal struc-tures of h-M340H-b4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9–2.0 A ̊ resolution showed that b4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest Km for the trisaccharide. Present studies suggest that b4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1, 3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan. Published by Elsevier Ltd.

Research paper thumbnail of Structure and catalytic cycle of beta-1,4-galactosyltransferase

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoc... more Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of Preparation and electrochemical validation of rGO-TiO2-MoO3 ternary nanocomposite for efficient supercapacitor electrode

Diamond and Related Materials

Research paper thumbnail of Graphene based ceria nanocomposite synthesized by hydrothermal method for enhanced supercapacitor performance

Diamond and Related Materials

Research paper thumbnail of Structure and catalytic cycle of �-1,4-galactosyltransferase

Curr Opin Struct Biol, 2004

β-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconj... more β-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of Oligosaccharide Preferences of b1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human b1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety

Research paper thumbnail of Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway

Journal of Molecular Biology, 2006

During the catalytic cycle of b1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn 2C fo... more During the catalytic cycle of b1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn 2C followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn 2C and Mn 2C -UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn 2C -UDP-GalNAc-Glc-a-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn 2C undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7 Å away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its nonhydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the bphosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the b-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Research paper thumbnail of Oligosaccharide Preferences of β1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human β1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety

Journal of Molecular Biology, 2005

Switzerland b-1,4-Galactosyltransferase-I (b4Gal-T1) transfers galactose from UDPgalactose to N-a... more Switzerland b-1,4-Galactosyltransferase-I (b4Gal-T1) transfers galactose from UDPgalactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is b-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of b4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human b4Gal-T1 (h-b4Gal-T1) and the mutant Met340His-b4Gal-T1 (h-M340H-b4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcb1,2-Mana1,6 (GlcNAcb1,2-Mana1,3)Man; the 1,6-arm trisaccharide, GlcNAcb1,2-Mana1,6-Manb-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcb1,2-Mana1,3-Manb-OR (1,2-1,3-arm) and GlcNAcb1,4-Mana1,3-Manb-OR (1,4-1,3-arm); and the trisaccharide GlcNAcb1,4-GlcNAcb1,4-GlcNAc (chitotriose). With the wild-type h-b4Gal-T1, the K m of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-b4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0 Å resolution showed that b4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K m for the trisaccharide. Present studies suggest that b4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1, 3-arm of a bi-or tri-antennary oligosaccharide chain of N-glycan.

Research paper thumbnail of Structure and catalytic cycle of β-1,4-galactosyltransferase

Current Opinion in Structural Biology, 2004

b-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconj... more b-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of The Role of Tryptophan 314 in the Conformational Changes of β1,4-Galactosyltransferase-I

Journal of Molecular Biology, 2003

b1,4-Galactosyltransferase-I (b4Gal-T1) undergoes critical conformational changes upon substrate ... more b1,4-Galactosyltransferase-I (b4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313 -316) and the long loop (residues 345 -365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of b4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, a-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I 0 ) (1.9 Å resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and a-lactalbumin, crystallizes in a conformation (2.3 Å resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme.

Research paper thumbnail of with a Pentasacc n

T1, the Km of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, an... more T1, the Km of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal struc-tures of h-M340H-b4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9–2.0 A ̊ resolution showed that b4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest Km for the trisaccharide. Present studies suggest that b4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1, 3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan. Published by Elsevier Ltd.

Research paper thumbnail of Structure and catalytic cycle of beta-1,4-galactosyltransferase

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoc... more Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of Preparation and electrochemical validation of rGO-TiO2-MoO3 ternary nanocomposite for efficient supercapacitor electrode

Diamond and Related Materials

Research paper thumbnail of Graphene based ceria nanocomposite synthesized by hydrothermal method for enhanced supercapacitor performance

Diamond and Related Materials

Research paper thumbnail of Structure and catalytic cycle of �-1,4-galactosyltransferase

Curr Opin Struct Biol, 2004

β-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconj... more β-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of Oligosaccharide Preferences of b1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human b1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety

Research paper thumbnail of Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway

Journal of Molecular Biology, 2006

During the catalytic cycle of b1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn 2C fo... more During the catalytic cycle of b1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn 2C followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn 2C and Mn 2C -UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn 2C -UDP-GalNAc-Glc-a-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn 2C undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7 Å away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its nonhydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the bphosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the b-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Research paper thumbnail of Oligosaccharide Preferences of β1,4-Galactosyltransferase-I: Crystal Structures of Met340His Mutant of Human β1,4-Galactosyltransferase-I with a Pentasaccharide and Trisaccharides of the N-Glycan Moiety

Journal of Molecular Biology, 2005

Switzerland b-1,4-Galactosyltransferase-I (b4Gal-T1) transfers galactose from UDPgalactose to N-a... more Switzerland b-1,4-Galactosyltransferase-I (b4Gal-T1) transfers galactose from UDPgalactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is b-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of b4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human b4Gal-T1 (h-b4Gal-T1) and the mutant Met340His-b4Gal-T1 (h-M340H-b4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcb1,2-Mana1,6 (GlcNAcb1,2-Mana1,3)Man; the 1,6-arm trisaccharide, GlcNAcb1,2-Mana1,6-Manb-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcb1,2-Mana1,3-Manb-OR (1,2-1,3-arm) and GlcNAcb1,4-Mana1,3-Manb-OR (1,4-1,3-arm); and the trisaccharide GlcNAcb1,4-GlcNAcb1,4-GlcNAc (chitotriose). With the wild-type h-b4Gal-T1, the K m of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-b4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0 Å resolution showed that b4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K m for the trisaccharide. Present studies suggest that b4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1, 3-arm of a bi-or tri-antennary oligosaccharide chain of N-glycan.

Research paper thumbnail of Structure and catalytic cycle of β-1,4-galactosyltransferase

Current Opinion in Structural Biology, 2004

b-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconj... more b-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Research paper thumbnail of The Role of Tryptophan 314 in the Conformational Changes of β1,4-Galactosyltransferase-I

Journal of Molecular Biology, 2003

b1,4-Galactosyltransferase-I (b4Gal-T1) undergoes critical conformational changes upon substrate ... more b1,4-Galactosyltransferase-I (b4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313 -316) and the long loop (residues 345 -365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of b4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, a-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I 0 ) (1.9 Å resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and a-lactalbumin, crystallizes in a conformation (2.3 Å resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme.