Abstract
D-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K(m)(Xu) = 24 ± 3 μm and k(cat) = 35 ± 5 s(-1). Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.
Highlights
D-Xylulokinase (XK), the final enzyme in the glucuronate-xylulose pathway, produces a key regulator of lipogenesis, xylulose 5-phosphate
XK may play an important role in metabolic disease, given that xylulose 5-phosphate (Xu5P) is a key regulator of glucose metabolism and lipogenesis
As xylitol is converted to Xu5P through the reactions, xylitol 3 D-xylulose 3 Xu5P within the GX pathway, these findings indicate that the pathway is constitutively active in vivo and can regulate hepatic carbohydrate and lipid metabolism through production of Xu5P
Summary
D-Xylulokinase (XK), the final enzyme in the glucuronate-xylulose pathway, produces a key regulator of lipogenesis, xylulose 5-phosphate. Excess intake of dietary glucose leads to the accumulation of lipid in skeletal muscle [2] This is closely linked with peripheral insulin resistance and if left untreated can result in type-2 diabetes mellitus and downstream vascular complications [2]. Peripheral insulin resistance in skeletal muscle further increases the conversion of dietary carbohydrate into triglyceride through lipogenesis in the liver [3]. In this scenario of increasing fat deposits, insulin resistance, and metabolic disease, therapies that inhibit lipogenesis are becoming increasingly attractive. A hitherto unexplored approach is to target production of the metabolite xylulose 5-phosphate (Xu5P), which regulates lipogenesis and glycolysis in the liver independently of insulin [4]. Obese ob/ob mice deficient in carbohydrate-response element-binding protein expression dis-
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