Dissection of alpha mannosidases: from reaction coordinate to inhibition

Sugars are more than just food. Sugars coat all our cells and the cells of our pathogens. The communication between cells, both healthy and pathogenic, both normal and tumour is governed by sugars. Sugars are 'the language of the cell'. The chemical factories that make, modify and tune sugars are called 'enzymes'. The control and organisation of these enzymatic machines is central to the healthy organism and, conversely, their subversion leads to changes in cell growth, auto immune disease and cancers. An unusual sugar, called mannose, is of particular interest to scientists as its chemistry is very difficult (its crowded centres make reactions difficult) and because the enzymes involved in its modification are both highly unusual and implicated in cell division and disease. The aim of this BBSRC grant is to study enzymes involved in mannose chemistry, to dissect them at the three-dimensional level and then to use cunning chemistry to both study their reaction and inspire and analyse inhibitors that should lead to chemical probes of healthy cells and potential therapeutic agents.

Mannosides are amongst the most interesting sugars in both chemistry and biology. Mannose chemistry, both enzymatic and in organic synthesis, is fundamentally extremely challenging; the axial O2 substituent preventing facile chemistry at the anomeric centre for steric reasons. Yet, despite these chemical challenges, mannoside biology is extremely important and nowhere more so than in the degradation and modification of the alpha mannosides present in human N-glycans. Glycan modification is central to the healthy cell whilst its subversion, to allow cancer-specific glycan extensions, is essential for metastatic progression and may also play a role in salvage pathways to evade (the otherwise powerful) glucosidase-based inhibition strategies against virus and cancer. Additionally, bacterial hydrolysis of human N-glycan alpha mannosides is a signature both of symbiotic human gut bacteria, which harness super-families of diverse alpha mannosidases during gut colonisation, and also a feature of streptoccal virulence. The work described herein aims to tackle the mechanistic enzymology of three different families of alpha-mannosidase all of whose substrates are human N-glycans. The work will probe the unusual reaction transition-states, aiming to resolve controversies about the reaction pathway of alpha mannosidase II homologs and help to inform the design of specific inhibitors for this medically relevant enzyme; both as potential therapeutic agents and cellular probes. The work will also embark on the analysis of two novel alpha mannosidase families which have previously had no structural, kinetic or mechanistic dissection. The goal will be to analyze the 3-D structures of mechanistically-insightful complexes and to use these, together with kinetic dissection of wild-type and variant enzymes, to inform inhibitor design. The work will provide the foundation for the future exploitation and inhibition of alpha mannosidases in cellular research