Structure and Function of Human Xylulokinase, an Enzyme with Important Roles in Carbohydrate Metabolism

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. Background: d-Xylulokinase (XK), the final enzyme in the glucuronate-xylulose pathway, produces a key regulator of lipogenesis, xylulose 5-phosphate. Results: The structure of human XK was determined, and its catalytic activity and inhibition were characterized. Conclusion: Human XK is selective for d-xylulose and is inhibited by 5-deoxy-5-fluoro-d-xylulose. Significance: Inhibition of XK could clarify its roles in sugar metabolism, lipogenesis, and metabolic disease.