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Title: Structural Snapshots of Beta- 1,4Galactosyltransferase-l Along the Kinetic Pathway

Abstract

During the catalytic cycle of {beta}1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn{sup 2+} 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{sup 2+} and Mn{sup 2+}-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn{sup 2+}-UDP-GalNAc-Glc-{alpha}-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn{sup 2+} 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 {angstrom} 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 non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structuremore » also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the {beta}-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the {beta}-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.« less

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930111
Report Number(s):
BNL-80755-2008-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US200822%%1324
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology; Journal Volume: 357
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; 36 MATERIALS SCIENCE; ATOMS; CATALYSIS; CATTLE; COMPLEXES; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; ENZYMES; HUMAN POPULATIONS; HYDROGEN; IONS; KINETICS; MOLECULES; MUTANTS; OXYGEN; RESOLUTION; ROTATION; WATER; national synchrotron light source

Citation Formats

Ramakrishnan,B., Ramasamy, V., and Qasba, P.. Structural Snapshots of Beta- 1,4Galactosyltransferase-l Along the Kinetic Pathway. United States: N. p., 2006. Web. doi:10.1016/j.jmb.2006.01.088.
Ramakrishnan,B., Ramasamy, V., & Qasba, P.. Structural Snapshots of Beta- 1,4Galactosyltransferase-l Along the Kinetic Pathway. United States. doi:10.1016/j.jmb.2006.01.088.
Ramakrishnan,B., Ramasamy, V., and Qasba, P.. Sun . "Structural Snapshots of Beta- 1,4Galactosyltransferase-l Along the Kinetic Pathway". United States. doi:10.1016/j.jmb.2006.01.088.
@article{osti_930111,
title = {Structural Snapshots of Beta- 1,4Galactosyltransferase-l Along the Kinetic Pathway},
author = {Ramakrishnan,B. and Ramasamy, V. and Qasba, P.},
abstractNote = {During the catalytic cycle of {beta}1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn{sup 2+} 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{sup 2+} and Mn{sup 2+}-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn{sup 2+}-UDP-GalNAc-Glc-{alpha}-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn{sup 2+} 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 {angstrom} 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 non-hydrogen 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 {beta}-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the {beta}-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.},
doi = {10.1016/j.jmb.2006.01.088},
journal = {Journal of Molecular Biology},
number = ,
volume = 357,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
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