An Investigation into β-Glucuronidases

Conjugated carbohydrates, such as proteoglycans, play pivotal roles in a number of diverse biological processes. Their structural complexity encodes information that regulates intercellular communication, recognition events and cellular activity. As such, the biosynthesis and catabolism of these complex molecules requires the tight regulation of a large number of enzymes. Two enzymes that are of particular interest in the metabolism of glycosaminoglycans are exo-β-glucuronidase and the endo-β-glucuronidase, heparanase. These two glycosidases play an important role in the catabolism of glycosaminoglycan chains from extracellular matrix and basement membrane proteoglycans. Over-expression of these enzymes has been reported in a number of tumours, and they have been implicated in tumour metastasis and angiogenesis. Thus, there is considerable interest in the characterisation of these enzymes for the development of novel enzyme inhibitors. The work described in this thesis is divided into two parts. The first part describes the cloning, expression and characterisation of a novel heparanase from the pathogenic bacterium Burkholderia pseudomallei. In addition to the enzyme's hydrolase activity against heparan sulfate, the B. pseudomallei heparanase was also found to have significant exo-β-glucuronidase activity. This is the first report of a bacterial enzyme that is capable of cleaving heparan sulfate via a hydrolase mechanism. In the second part of the work, the X-ray crystal structure of human β-glucuronidase was used to design novel enzyme inhibitors. A number of computational tools such as GRID, molecular docking and de novo ligand design were employed to generate a library of substituents that could be introduced at the C-1, C-2 and/or C-3 positions of specific carbohydrate templates that would potentially improve ligand binding to the enzyme. A number of 1-thioglucuronides, with substituted benzylic or aliphatic aglycon moieties, identified by the structure-based ligand design process, were chosen for ...