Enzymes Associated with the Complications of Diabetes Mellitus

Abstract

Diabetes mellitus (DM) is a metabolic disease resulting from failures in the production or response to the hormone insulin. Much of the pathogenesis and mortality attributed to DM are due to the long-term complications of hyperglycaemia, which is characteristic of the disease. This thesis presents structural and functional studies of two previously uncharacterised human enzymes, dihydrodipicolinate synthase-like protein (DHDPSL) and D-xylulokinase (XK). Both enzymes were revealed to have unexplored associations with DM. DHDPSL is distantly related (~25% sequence identity) to a family of Schiff base-dependent aldolases that include dihydrodipicolinate synthase and N-acetylneuraminate lyase. Despite these distant homologies the biological function of DHDPSL is unknown. It also does not map to any known metabolic pathway in humans, but is targeted to the mitochondrial compartment consistent with the presence of a mitochondrial targeting sequence. There are also strong associations between mutations in the Dhdpsl gene and primary hyperoxaluria type III a rare disorder of endogenous oxalate production. The DHDPSL crystal structure was determined by X-ray crystallography utilising in situ proteolysis of a fusion of DHDPSL with maltose-binding protein for crystallisation. Two apoforms and six Schiff base complexes with potential ligands were analysed at best to 2.0 Å resolution and with an Rfree of 18.3%. DHDPSL is folded as (a/ss)₈-barrel with a C-terminal subdomain and forms a tetramer in the crystal. The structural consequences of the diseaserelevant DHDPSL mutations were analysed and were found to largely affect the C-terminal subdomain. Findings also showed that DHDPSL acts as an oxaloacetate decarboxylase and is therefore likely to be a bifunctional oxaloacetate decarboxylase /4-hydroxy-2-ketoglutarate aldolase present in the liver and kidney mitochondria. Overall, these results revealed the presence of a potentially significant metabolic pathway in mitochondria whereby oxaloacetate can be converted to pyruvate. XK has a potential role in the regulation of de novo lipogenesis in the liver that has gained little previous attention. Excessive hepatic lipid accumulation is linked to impaired insulin response and the development of DM. XK was identified and produced recombinantly in Eschericia coli aided by molecular chaperones. Crystals suitable for structural analysis were obtained after five generations of repeated seeding. Five crystal structures were used to analyse substrate binding, the best of which was determined at 2.0 Å resolution with an Rfree of 17.8%. XK assumes an actin-like two-domain FGGY sugar kinase fold. The most striking feature revealed by the XK molecular structure was a dramatic domain movement that must accompany catalysis. A competitive inhibitor of XK, 5-deoxy-5-fluoro-D-xylulose (Ki of 25.4 M) was also functionally validated and structurally analysed. A supplemental structural study of a major hypoxic response protein of unknown function, Rv1738, from the causative agent of tuberculosis, Mycobacterium tuberculosis is also described. This study presents the first novel protein structure to be determined by the racemic protein crystallography method. The Rv1738 structure exposes a relationship with a family of ribosome-inhibiting stress-response proteins that may be indicative of its function.