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Structural Studies of Glycosyl Hydrolases

This major area of research involves enzymes that recognize and act upon carbohydrates, including especially glycosidases involved in the protein glycosylation pathway.

Golgi alpha-mannosidase II
Insights from these results are now being applied to enzymes in the mammalian glycosylation pathway. A key component in this pathway, Golgi alpha-mannosidase II, is a target for inhibition by compounds that can decrease the size and aggressiveness of many tumour types. We have solved the structure of the Drosophila homologue as a model to study the interaction of known inhibitors and the design of new inhibitors to a-mannosidase II. This structure of the glycosyl hydrolase family 38 enzyme displays a novel folding pattern and acts through a fascinating catalytic process that allows 2 glycosidic bonds to be cleaved successively in the same catalytic site.

Lysosomal alpha-mannosidase

Related to the Golgi enzyme, inhibition of the lysosomal Family 38 mannosidase is thought ot contribute to the side effects of clinical trials of Golgi mannosidase inhibitors. We have produced the lysosomal enzyme in our Drosophila system and are analyzing its enzymatic and inhibitory properties. We are also working towards the structural analysis of this homologue.

Intestinal Maltase-Glucoamylase (MGA)
MGA is involved in starch breakdown in mammalian cells. Inhibition of MGA and other alpha-glucosidases is proposed to be a novel approach to treatment of Type II Diabetes. We have expressed this Family GH31 enzyme in Drosophila cells and studied the activities of a series of specific inhibitors under development as anti-diabetics. We are preparing a full structure determination of this enzyme and hope to extend this work to others in the starch processing pathway.

Cellumonas fimi beta-1,4-glycanase
For several years, we have studied the recognition and enzymatic mechanism of a bacterial beta-1,4-glycanase by determining the structure to high resolution of the enzyme and its complexes with substrate analogues that can be trapped in the active site. The complexes represent covalent intermediates in the catalytic mechanism and have led to a number of insights into the details of how this class of enzymes works. More recently, we have examined complexes with analogues of aspects of the putative transition state of the reaction.

Carbohydrate binding modules
Cellulose-binding domains (CBDs), or carbohydrate-binding modules (CBMs), are frequently linked to glycosyl hydrolase catalytic domains in the mature enzymes of bacteria and fungi. CBMs have been assigned to a number of families based on sequence analyses. We have determined the structures of CBMs from a number of families by X-ray crystallography and we are identifying their sugar binding regions by analyzing saccharide complexes.

Golgi alpha-mannosidase II