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A structural and thermodynamic comparison of substrate interactions and catalysis by family 6 glycosyltransferases from Bacteroides ovatus, Parachlamydia acanthamoebae, and Bos taurus

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Date Issued:
2018
Abstract/Description:
Family 6 Glycosyltransferases (GT6s) are involved in the biosynthesis of complex glycans and can be found in all vertebrates, cyanophages, and some bacteria and unicellular eukaryotes. Understanding variations within family 6 GTs is important because of the roles of their products in cellular recognition, intercellular interactions, pathogenicity, and immunity and is likewise important for understanding the evolution of GTs. PaGT6 (from Parchlamydia acanthamoebae) and α3GT (from Bos taurus) both require a divalent metal ion for catalysis which binds to a DXD motif. In BoGT6a from Bacteroides ovatus a NXN motif replaces DXD, and activity is metal-independent. However, mutating the NXN motif in BoGT6a to DXD did not introduce metal-dependency, indicating that metal-dependency is linked to additional differences. Calorimetric studies have shown that the presence of a divalent metal ion enhances UDP and donor substrate binding to PaGT6 and causes an increase in the entropy of the interaction. Protein modelling of PaGT6 has revealed that the presence of Mn2+ allows a hydrogen bond to form between Asp 97 and UDP-GalNAc, causing the donor substrate to bend and form hydrogen bonds with His 119, Asn 229, Lys 228, and Arg 234. These interactions do not occur in the absence of Mn2+. Investigation of acceptor substrate binding revealed that the presence of UDP enhances acceptor substrate binding to BoGT6a and PaGT6. Calorimetric titrations of BoGT6a with 2-fucosyllactose in the absence and presence of UDP showed that UDP increases the affinity of 2-fucosyllactose 16-fold with little effect on ΔH. Measurements of ΔCp for 2-fucosyllactose binding indicate that there is not a hydrophobic effect for the binding of 2-fucosyllactose. The preferred acceptor substrate for the bovine and Bacteroides GT6 has a β-1,4 linked galactose, but P. acanthamoebae GT6 prefers an acceptor substrate with a β-1,3 linked galactose. The N-terminus of the catalytic domain of bacterial GT6s is truncated by 47 residues relative to the catalytic domain of bovine α3GT. Removal of this region from α3GT results in an unfolded protein, indicating that although this region is not directly involved in substrate binding, it forms interactions necessary for the stability of the catalytic domain.
Title: A structural and thermodynamic comparison of substrate interactions and catalysis by family 6 glycosyltransferases from Bacteroides ovatus, Parachlamydia acanthamoebae, and Bos taurus.
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Name(s): Stinson, Brittany, author
Brew, Keith, Thesis advisor
Florida Atlantic University, Degree grantor
Charles E. Schmidt College of Science
Department of Biological Sciences
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Created: 2018
Date Issued: 2018
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 136 p.
Language(s): English
Abstract/Description: Family 6 Glycosyltransferases (GT6s) are involved in the biosynthesis of complex glycans and can be found in all vertebrates, cyanophages, and some bacteria and unicellular eukaryotes. Understanding variations within family 6 GTs is important because of the roles of their products in cellular recognition, intercellular interactions, pathogenicity, and immunity and is likewise important for understanding the evolution of GTs. PaGT6 (from Parchlamydia acanthamoebae) and α3GT (from Bos taurus) both require a divalent metal ion for catalysis which binds to a DXD motif. In BoGT6a from Bacteroides ovatus a NXN motif replaces DXD, and activity is metal-independent. However, mutating the NXN motif in BoGT6a to DXD did not introduce metal-dependency, indicating that metal-dependency is linked to additional differences. Calorimetric studies have shown that the presence of a divalent metal ion enhances UDP and donor substrate binding to PaGT6 and causes an increase in the entropy of the interaction. Protein modelling of PaGT6 has revealed that the presence of Mn2+ allows a hydrogen bond to form between Asp 97 and UDP-GalNAc, causing the donor substrate to bend and form hydrogen bonds with His 119, Asn 229, Lys 228, and Arg 234. These interactions do not occur in the absence of Mn2+. Investigation of acceptor substrate binding revealed that the presence of UDP enhances acceptor substrate binding to BoGT6a and PaGT6. Calorimetric titrations of BoGT6a with 2-fucosyllactose in the absence and presence of UDP showed that UDP increases the affinity of 2-fucosyllactose 16-fold with little effect on ΔH. Measurements of ΔCp for 2-fucosyllactose binding indicate that there is not a hydrophobic effect for the binding of 2-fucosyllactose. The preferred acceptor substrate for the bovine and Bacteroides GT6 has a β-1,4 linked galactose, but P. acanthamoebae GT6 prefers an acceptor substrate with a β-1,3 linked galactose. The N-terminus of the catalytic domain of bacterial GT6s is truncated by 47 residues relative to the catalytic domain of bovine α3GT. Removal of this region from α3GT results in an unfolded protein, indicating that although this region is not directly involved in substrate binding, it forms interactions necessary for the stability of the catalytic domain.
Identifier: FA00013047 (IID)
Degree granted: Dissertation (Ph.D.)--Florida Atlantic University, 2018.
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Includes bibliography.
Subject(s): Glycosyltransferases
Bacteroides
Chlamydiales
Acanthamoeba
Bos taurus
Cattle
Held by: Florida Atlantic University Libraries
Sublocation: Digital Library
Persistent Link to This Record: http://purl.flvc.org/fau/fd/FA00013047
Use and Reproduction: Copyright © is held by the author, with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Use and Reproduction: http://rightsstatements.org/vocab/InC/1.0/
Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.