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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
Başlık:
A Structural and Thermodynamic Comparison of Substrate Interactions and Catalysis by Family 6 Glycosyltransferases from Bacteroides Ovatus, Parachlamydia acanthamoebae, and Bos Taurus
Yazar:
Stinson, Brittany, author. (orcid)0000-0002-4853-5608
ISBN:
9780438013803
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (136 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Keith Brew Committee members: Zhongwei Li; Michelle Lizotte-Waniewski; Ewa Wojcikiewicz.
Özet:
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 alpha3GT (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 ?H. Measurements of ?Cp 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 ?-1,4 linked galactose, but P. acanthamoebae GT6 prefers an acceptor substrate with a ?-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 ?3GT. Removal of this region from ?3GT 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.
Notlar:
School code: 0119
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(681561.1) | 681561-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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