Award details

Screening Carbohydrate-Active Enzymes - a Fluorescent Solution

Reference	BBS/E/J/000CA425
Principal Investigator / Supervisor	Dr Rob Field
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	John Innes Centre Department
Funding type	Research
Value (£)	5,163
Status	Completed
Type	Institute Project
Start date	01/10/2010
End date	30/09/2011
Duration	12 months

Abstract

WHY ARE NEW GLYCOSYLTRANSFERASE (GT) ASSAYS NEEDED? With our current level of understanding, it is only possible to use genome sequence information to identify a putative GT, but not to predict which sugar it transfers (donor specificity) or which sugar it transfers onto (acceptor specificity). The most common GT assay format is based on radiolabelled sugar-nucleotides and follows the transfer of radiolabel onto the acceptor. The requirement for said radiochemical material is frequently a limiting factor; even in those cases where the acceptor is known, its availability is frequently limited - GT acceptors are usually structurally complex carbohydrates which are not commercially available and whose synthetic preparation is cumbersome. The development of alternative assay formats which do not require access to the acceptor substrate and/or radiolabelled donor is therefore of considerable scientific importance. The availability of such assay formats will allow organism-wide studies into the substrate specificity and biological function of GTs. Aims & objectives. The main aim of this project is the development and biological application of novel, 'colour-coded' sugar-nucleotides, in which each individual sugar is linked to a unique fluorophore , allowing selective detection e.g. in enzyme mixtures, cell extracts etc. Specifically, we aim to prepare a set of 'colour-coded' derivatives of four common UDP-sugars (Gal, Glc, GlcNAc, GalNAc) and two TDP-sugars (Glc and 6-deoxy-D-xylo-4-hexulose). Work programme. This project includes three main activities: (i) the development of novel, fluorescent and spectroscopically unique sugar-nucleotides (e.g. with individual colours) derived from lead compound 1 (ii) the in vitro characterisation of the novel sugar-nucleotides against a panel of GTs and other sugar-nucleotide-dependent enzymes (individual enzymes) (iii) application of the fluorophores for the analysis of enzyme mixtures / cell extracts /fractions

Summary

unavailable

Committee	Not funded via Committee
Research Topics	Plant Science, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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