Dissecting the mechanism by which glycosyltransferases catalyse mannosyl transfer
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
The linking of sugars to a variety of different proteins has an important influence on the function of cells. The biological catalysts or enzymes that speed up the reactions in which sugars are linked to other molecules are known as glycosyltransferases. Although these enzymes and are industrially and biologically important, they have not been extensively studied because they are difficult to produce in large amounts. In this project we will take advantage of our ability to produce significant quantities of glycosyltransferases that catalyse the transfer of the sugar mannose onto other molecules. The three dimensional structure of these enzymes will be determined and the information will be used to synthesise inhibitors of these biological catalysts and to use molecular engineering techniques to manipulate their biological properties.
Technical Summary
Although mannose-containing polymers are widespread in nature, there is a paucity of structural and mechanistic information on the enzymes that catalyze mannosyltransfer. Recent studies by our three groups [Flint et al. (2005) Nat. Struct. Mol. Biol. 12, 608-14] have begun to unravel the structural basis for the catalytic activity and plasticity of substrate recognition of the retaining GDP-Man transferase, mannosylglycerate synthase, which lays a foundation upon which to dissect mannosyl transfer. This application will build upon our studies on mannosylglycerate synthase and a significant additional body of preliminary data, of both retaining and inverting mannosyltransferases (including one of the key enzymes of glycobiology, dolichyl-phosphate -D-mannose synthase) to dissect the mechanism of action and specificity of mannosyltransferases. This will underpin the engineering of these enzymes to increase their utility as biosynthetic tools, underpin novel therapeutic strategies that target glycan decoration and, and, through the modulation of key enzymes that catalyse mannosyltransfer, provide profound insights into cellular function To date, there are no known selective inhibitors of any retaining glycosyltransferases with sufficient potency to allow modulation of the function of these enzymes in vivo. This void has hampered not only the understanding of the role of mannose decoration in biology, but also the exploitation of mannosyl transfer in drug design. The key goals that will be addressed in this project are: (a) Determine how structure dictates specificity and the mechanism of catalysis of mannosyltransferases (b) Exploitation of such information in the design of new enzyme inhibitors which reflect both structural and mechanistic features (c) Interrogation of the evolution of the mechanisms of mannosyl transfer, and its exploitation in the development of novel biocatalysts.
People |
ORCID iD |
David Bolam (Principal Investigator) |
Publications
Cartmell A
(2008)
The Cellvibrio japonicus mannanase CjMan26C displays a unique exo-mode of action that is conferred by subtle changes to the distal region of the active site.
in The Journal of biological chemistry
Nielsen MM
(2011)
Substrate and metal ion promiscuity in mannosylglycerate synthase.
in The Journal of biological chemistry
Offen WA
(2009)
Structure of the Michaelis complex of beta-mannosidase, Man2A, provides insight into the conformational itinerary of mannoside hydrolysis.
in Chemical communications (Cambridge, England)
Suits MD
(2010)
Structure and kinetic investigation of Streptococcus pyogenes family GH38 alpha-mannosidase.
in PloS one
Tailford LE
(2009)
Understanding how diverse beta-mannanases recognize heterogeneous substrates.
in Biochemistry
Tailford LE
(2008)
Structural and biochemical evidence for a boat-like transition state in beta-mannosidases.
in Nature chemical biology
Zhu Y
(2010)
Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont.
in Nature chemical biology
Description | We showed that mannosylglycerate synthase, unusually for a glycosyltransferase, displays a preference for group II metals such as magnesium and calcium, over trace metals such as manganese, nickel and cobalt. Mutation of the key coordinating Histidine abolishes the ability of the enzyme to use group II metals but increases kcat when trace elements are used. The structure of the enzyme shows that the histidine is on a mobile loop that contributes to both donor and acceptor binding. A combination of biochemical and structural data, together with the altered metal preference, provides novel insight into the mechanism by which metals contribute to the action of glycosyltransferases. |
Exploitation Route | Understanding the detailed mechanism of mannose transfer has implications for our understanding of several serious congenital diseases and could also underpin development of novel glycoconjugates for a range of applications (e.g. medical, biotech). |
Sectors | Chemicals,Pharmaceuticals and Medical Biotechnology |
Description | It is not clear that there have been any non-academic impacts from this grant. |
Impact Types | Economic |