Journal of Molecular Biology
Volume 365, Issue 3, 19 January 2007, Pages 783-798
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Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli

https://doi.org/10.1016/j.jmb.2006.10.068Get rights and content

Abstract

The primary metabolic route for d-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates d-xylulose, the last step in this conversion. The apo and d-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 Å resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which d-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.

Introduction

The ability of an organism to metabolize d-xylose and some other pentoses is conferred by their conversion to d-xylulose-5-phosphate (X5P), a compound that is able to enter the non-oxidative branch of the pentose phosphate pathway. In eukaryotes capable of assimilating xylose, X5P is produced through the action of xylose reductase, xylitol dehydrogenase and xylulose kinase (XK; EC 2.7.1.17). The first two enzymes are replaced in prokaryotes with xylose isomerase, which interconverts xylose and xylulose directly. There has been much interest in metabolic engineering of the two pathway variants for the past two decades because of the possibility of fermenting the vast quantities of xylose in agricultural waste products to produce ethanol.1,2 These prospects have driven the study of the catalytic properties and substrate specificities of xylose reductase, xylitol dehydrogenase and xylose isomerase from many different sources. XK has been less well studied.

Physiological studies have shown that XK is essential for growth on xylose or xylulose and may limit the overall rate of pentose sugar utilization.3 In addition to its role in xylose metabolic assimilation, its activity with the alternative substrate 1-deoxy-d-xylulose4 implicates it in the biosynthesis of terpenoids,5 thiamine6 and pyridoxal.7 The enzyme from several prokaryotes and lower eukaryotes, including Escherichia coli, yeasts and fungi, has been studied and XK activity has been identified in higher eukaryotes.3,8,9

On the basis of amino acid sequence similarity, the 53 kDa E. coli XK (ecXK), encoded by the xylB gene contains an ATPase fingerprint consisting of five conserved regions found in a large group of proteins, including sugar kinases, actins, and heat shock proteins.10 Structurally, superfamily members consist of two domains, I and II, which are separated by a cleft forming the active site. Generally, members bind ATP and catalyze the hydrolysis of the γ-phosphate group or its transfer to a substrate such as a sugar hydroxyl group. Catalysis is preceded by a domain closure that is induced by substrate binding, as exemplified by the induced-fit mechanism of yeast hexokinase.11 Phospho-transfer is promoted by two highly conserved aspartate residues. One is located in the N-terminal region of domain I and interacts with the ATP-associated Mg2+. It is invariant across superfamily members and belongs to a signature sequence that identifies them.12 The second aspartate appears to function as a general base, activating the nucleophile for attack.

When the entire sequence is examined, ecXK is most similar to a family of carbohydrate kinases phosphorylating fucose, glucose, glycerol and xylulose. Of these, X-ray crystal structures of glycerol kinase (GK) from E. coli (ecGK) and Enterococcus casseliflavus have been reported, and detailed relationships between structure and function have been determined for GK.13,14 Like many other members of the family, the activity of GK is regulated by binding of small ligands (fructose-1,6-bisphosphate) as well as by interactions with other proteins (the glucose-specific phosphocarrier protein IIIGlc).15 The enzyme from E. casseliflavus can be covalently phosphorylated at His232, resulting in a substantial activation.14

Details surrounding XK kinetic and structural properties have often been inferred from these related enzymes. These include a substrate-induced conformational change creating a high-affinity ATP-binding site that has been implied but never observed in the carbohydrate kinases, and which has kinetic consequences.16,17 The oligomeric state of XK is unclear but important, since other family members such as ecGK can be regulated by effector-modulated oligomerization. The kinetic mechanism of XK has not been established, and precedents within the family are mixed, with some members binding substrates in an ordered manner while others are random. A comprehensive quantitative evaluation of substrate specificity for the enzyme has not been done. To shed light on these and other mechanistic issues, a combined structural and kinetic analysis of XK from E. coli is described here.

Section snippets

Overall structure

The structure of ecXK in the apo form has been determined at 2.7 Å resolution using multiple isomorphous replacement phasing. The apo structure was used to phase a xylulose-bound structure at 2.1 Å. This model consists of two protein molecules (A and B) that form the asymmetric unit, each consisting of residues 1–334 and 343–484 of the 484 predicted. There was no electron density corresponding to the region 335–342. There are also 310 water molecules that are observed in the asymmetric unit.

Materials

d-Xylulose (≥95% pure) was produced by microbial oxidation of d-arabitol.33 5-F-d-Xylulose was synthesized as described,34 and kindly provided by Professor Arnold Stütz from the Institute of Organic Chemistry, Graz University of Technology. All other reagents were from Sigma or Merck.

Cloning, protein expression and purification

The ecXK gene (xylB) was PCR-amplified from E. coli genomic DNA using the primers 5′-GCTAGTCCATATGTATATCGGGATAGATCTT-3′ and 5′-ACTGCCCGGGCGCCATTAATGGCAGAAGTTG-5′ (NdeI and SmaI sites are underlined). The resulting

Acknowledgements

Initial crystallization of ecXK by Youzhong Wan is gratefully acknowledged. This work was supported by the National Institutes of Health (GM66135 to D.K.W.), the Austrian Science Funds (FWF projects 15208 and 18275 to B.N.) and the Keck Foundation.

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