title: Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism.
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.
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Bunker
authors.forename: Esther M M
authors.initials: EM
authors.lastname: Bulloch
authors.forename: James M J
authors.initials: JM
authors.lastname: Dickson
authors.forename: Kerry M
authors.initials: KM
authors.lastname: Loomes
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014996:Xylulose / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D010428:Pentosephosphates; D011994:Recombinant Proteins; D014996:Xylulose; C031625:xylulose-5-phosphate; D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase