Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism - PubMed (original) (raw)

Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism

Richard D Bunker et al. J Biol Chem. 2013.

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.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

Three-dimensional structure and folding of hXK. A, molecule is shown as a ribbon diagram, in stereo, colored in rainbow style from the N terminus (dark blue) to the C terminus (red). The binding sites for the two substrates are indicated, with

d

-xylulose shown in orange and the nucleotide (modeled as AMP) shown in magenta, both in stick mode. B, topology diagram, with secondary structural elements colored as in A and labeled.

FIGURE 2.

FIGURE 2.

Analysis of the d-xylulose 5-kinase reaction catalyzed by hXK, using 1H NMR spectroscopy. _A,_1H NMR spectrum of an assay solution containing ATP and

d

-xylulose in the absence of hXK; B, after incubation with hXK. C, 1H NMR spectrum of xylulose 5-phosphate.

FIGURE 3.

FIGURE 3.

Kinetic analysis of the hXK reaction. A, relationship between initial reaction velocity and

d

-xylulose concentration in a single hXK activity assay. B, effect of the competitive inhibitor 5FX on the initial reaction velocity as a function of

d

-xylulose concentration. The assays were carried out with 600 m

m

ATP and 1.6 n

m

hXK and with varied

d

-xylulose and 5FX concentrations.

FIGURE 4.

FIGURE 4.

Binding of the substrate d-xylulose to hXK. A, stereo view of

d

-xylulose (Xu) modeled into its electron density. The electron density is from the initial 2m_Fo_ − D_Fc_ electron density map (blue mesh), calculated before Xu was included in the model, and is contoured at 1.0 σ. Hydrogen bonds are shown as broken lines, with distances in Å. The water molecule that is a consistent feature of the binding site is shown as a red sphere. B, fit of Xu into its binding site on domain-I, shown as a molecular surface and colored by chain succession as in Fig. 1. C, competitive inhibitor 5FX modeled into the initial 2m_Fo_ − D_Fc_ electron density map (blue mesh), calculated before it was included in the model, and contoured at 1.0 σ. The binding mode is virtually identical to that of Xu.

FIGURE 5.

FIGURE 5.

Nucleotide binding to hXK. Stereo view showing ADP (modeled as AMP) fitted to the 2m_Fo_ − D_Fc_ electron density map (blue mesh) calculated by BUSTER-TNT prior to inclusion of the ligand in the model and contoured at 1.0 σ. Relevant side chains (gray) and α-helices (yellow and orange) are labeled, and hydrogen bonds are drawn as dashed lines and distances shown in Å. A bridging water molecule is shown with a red sphere.

FIGURE 6.

FIGURE 6.

Modeling the closed form of hXK. The experimentally determined open form of hXK is shown in blue, superimposed on a modeled closed form of hXK in green, generated with Chimera (44), and based on a closed E. coli glycerol kinase structure (PDB code 1glj, see Ref. 43). The hinge axis, indicated by the purple circle, is normal to the page.

References

    1. Home P. D., Pacini G. (2008) Hepatic dysfunction and insulin insensitivity in type 2 diabetes mellitus. A critical target for insulin-sensitizing agents. Diabetes Obes. Metab. 10, 699–718 - PubMed
    1. McGarry J. D. (2002) Banting lecture 2001. Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes 51, 7–18 - PubMed
    1. Petersen K. F., Dufour S., Savage D. B., Bilz S., Solomon G., Yonemitsu S., Cline G. W., Befroy D., Zemany L., Kahn B. B., Papademetris X., Rothman D. L., Shulman G. I. (2007) The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome. Proc. Natl. Acad. Sci. U.S.A. 104, 12587–12594 - PMC - PubMed
    1. Uyeda K., Repa J. (2006) Carbohydrate-response element-binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis. Cell Metab. 4, 107–110 - PubMed
    1. Kabashima T., Kawaguchi T., Wadzinski B. E., Uyeda K. (2003) Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver. Proc. Natl. Acad. Sci. U.S.A. 100, 5107–5112 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources