Journal of Molecular Biology
Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli
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.
References (55)
- et al.
The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms
Chem. Biol.
(1998) - et al.
The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae
FEMS Microbiol Letters
(1998) - et al.
Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase. A microcalorimetric study
J. Biol. Chem.
(2005) - et al.
The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action
J. Biol. Chem.
(2000) - et al.
Crystal structures of an NAD kinase from Archaeoglobus fulgidus in complex with ATP, NAD, or NADP
J. Mol. Biol.
(2005) - et al.
Glycerol kinase from Escherichia coli and an Ala65 → Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation
Structure
(1998) - et al.
Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase
Arch. Biochem. Biophys.
(2001) - et al.
Structure of a complex between yeast hexokinase A and glucose. II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer
J. Mol. Biol.
(1980) - et al.
Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase
Structure
(2004) - et al.
Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily
Arch. Biochem. Biophys.
(1998)
Processing of X-Ray diffraction data collected in oscillation mode
Methods Enzymol.
X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase
Biochem. Biophys. Res. Commun.
Protein modelling for all
Trends Biochem. Sci.
EMAN: semiautomated software for high-resolution single-particle reconstructions
J. Struct. Biol.
SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields
J. Struct. Biol.
Similarity measures between images
Ultramicroscopy
Metabolic engineering of Saccharomyces cerevisiae for xylose utilization
Advan. Biochem. Eng. Biotechnol.
Metabolic engineering for improved fermentation of pentoses by yeasts
Appl. Microbiol. Biotechnol.
Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli
Appl. Environ. Microbiol.
Phosphorylation of 1-deoxy-d-xylulose by d-xylulokinase of Escherichia coli
Eur. J. Biochem.
1-deoxy-d-threo-2-pentulose: the precursor of the five-carbon chain of the thiazole of thiamine
J. Am. Chem. Soc.
Biosynthesis of vitamin B6: incorporation of d-1-deoxyxylulose
J. Am. Chem. Soc.
Molecular cloning of XYL3 (d-xylulokinase) from Pichia stipitis and characterization of its physiological function
Appl. Environ. Microbiol.
An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins
Proc. Natl Acad. Sci. USA
Glucose-induced conformational change in yeast hexokinase
Proc. Natl Acad. Sci. USA
Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein
Proc. Natl Acad. Sci. USA
Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase
Science
Cited by (39)
Binding interactions in a kinase active site modulate background ATP hydrolysis
2022, Biochimica et Biophysica Acta - Proteins and ProteomicsGlycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity
2017, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Aside from the reversibility and the lack of sensitivity to the usual regulatory mechanisms highlighted above, the reverse reaction of African trypanosomal GK has been proposed to proceed via a two-step mechanism in which the enzyme is first transiently autophosphorylated by G3P before the binding of ADP and the phospho group transfer from the phosphorylated residue to this acceptor substrate [15]. The reverse reaction mechanistically differs from the reactions of other FGGY kinase family members, which are basically forward reactions, and where both substrates (ATP and the respective phospho group acceptors) bind the enzymes (ternary complex formation) for direct transphosphorylation of the substrates [8,16–18]. From the unique mechanism of the reverse reaction of the trypanosomal GK, we envisaged that in the absence of ADP, the GK molecule might not be “locked-up” in the autophosphorylated state after dephosphorylating G3P, but may transfer the phospho group to water molecules, hence is forced to operate as a phosphatase (Scheme 1B).
Coupling xylitol dehydrogenase with NADH oxidase improves L-xylulose production in Escherichia coli culture
2017, Enzyme and Microbial TechnologyStructure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism
2013, Journal of Biological ChemistryCitation Excerpt :The photometric assay was also used to validate 5FX as an inhibitor of hXK (Fig. 3B). This compound has been shown to be a dead-end competitive inhibitor of ecXK and was used by Di Luccio et al. (16) to deduce the preferred substrate binding order for this enzyme. For hXK, the value of Ki for 5FX was 25 ± 2 μm, comparable with the Km for Xu.
D-Xylulose kinase from Saccharomyces cerevisiae: Isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli
2011, Protein Expression and PurificationCitation Excerpt :Using LC-MS/MS analysis of the respective peptide mixtures, we were able to identify 170 distinct peptides and thus obtained an overall sequence coverage of 92%. Using sequence similarity, Asp28 and Asp299 are positional homologs of the major catalytic residues, Asp6 and Asp233, in E. coli XK [27] and other members of the FGGY carbohydrate kinase family [28]. The peptides containing Asp28 and Asp299 were clearly identified by peptide sequencing of XKS1-Strep.
Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae
2011, Metabolic EngineeringCitation Excerpt :XI activity was determined using sorbitol dehydrogenase as previously described (Kuyper et al., 2003) at 30 °C. XK activity was measured by coupling ADP production to NADH oxidation via pyruvate kinase as earlier described (Di Luccio et al., 2007). Variations of XK activity, when compared to previous studies (Karhumaa et al., 2005), may be attributed to the fact that the XK assay is usually not performed at Vmax, due to the cost of pure xylulose.
- †
E.D.L., B.P. and J.V. made equal contributions to this work.