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Crystal Structure of Transaldolase B from Escherichia Coli Suggests A View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Article 715 Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the a/b barrel within the class I aldolase family Jia Jia1, Weijun Huang1, Ulrich Schörken2, Hermann Sahm2, Georg A Sprenger2, Ylva Lindqvist1* and Gunter Schneider1* Background: Transaldolase is one of the enzymes in the non-oxidative branch of Addresses: 1Division of Molecular Structural the pentose phosphate pathway. It transfers a C3 ketol fragment from a ketose Biology, Department of Medical Biochemistry and donor to an aldose acceptor. Transaldolase, together with transketolase, creates a Biophysics, Karolinska Institute, Doktorsringen 4, S-171 77 Stockholm, Sweden and 2Institut für reversible link between the pentose phosphate pathway and glycolysis. The Biotechnologie 1, Forschungszentrum Jülich enzyme is of considerable interest as a catalyst in stereospecific organic synthesis GmbH, PO Box 1913, D-52425 Jülich, Germany. and the aim of this work was to reveal the molecular architecture of transaldolase and provide insights into the structural basis of the enzymatic mechanism. *Corresponding authors. E-mail: YL, [email protected]; GS, [email protected] Results: The three-dimensional (3D) structure of recombinant transaldolase B from E. coli was determined at 1.87 Å resolution. The enzyme subunit consists of Key words: enzyme mechanism, evolution, gene a single eight-stranded a/b-barrel domain. Two subunits form a dimer related by permutation, protein crystallography, transaldolase a twofold symmetry axis. The active-site residue Lys132 which forms a Schiff Received: 14 Mar 1996 base with the substrate is located at the bottom of the active-site cleft. Revisions requested: 4 April 1996 Revisions received: 12 April 1996 Conclusions: The 3D structure of transaldolase is similar to structures of other Accepted: 16 April 1996 enzymes in the class I aldolase family. Comparison of these structures suggests Structure 15 June 1996, 4:715–724 that a circular permutation of the protein sequence might have occurred in transaldolase, which nevertheless results in a similar 3D structure. This © Current Biology Ltd ISSN 0969-2126 observation provides evidence for a naturally occurring circular permutation in an a/b-barrel protein. It appears that such genetic permutations occur more frequently during evolution than was previously thought. Introduction fructose-6-phosphate, to erythrose-4-phosphate yielding The pentose phosphate pathway for the metabolism of sedoheptulose-7-phosphate and glyceraldehyde-3-phos- glucose-6-phosphate can be described as containing three phate. Catalysis by transaldolase proceeds through the for- rather distinct enzyme systems [1]. One of these systems mation of a covalent intermediate, a Schiff base between catalyses a dehydrogenase/decarboxylase step which an active-site lysine residue and the dihydroxyacetone decarboxylates glucose-6-phosphate resulting in the for- moiety [2], similar to the mechanism of other class I mation of ribulose-5-phosphate and CO2 with concomitant aldolases (Fig. 1). The subunit of transaldolase from E. coli reduction of NADP+. The pathway includes an isomeriz- has a molecular weight of 35 kDa and the enzyme forms a ing system which produces an equilibrium mixture of dimer in solution [3] as do the enzymes from Saccharomyces ribose-5-phosphate, ribulose-5-phosphate and xylulose- cerevisiae [4] and Candida utilis [5]. The gene for transal- 5-phosphate. Finally, the pathway contains a sugar dolase B from E. coli codes for 317 amino acids, but the rearrangement system which uses the enzymes transketo- N-terminal methionine residue is cleaved off in the mature lase and transaldolase to produce three to seven carbon protein [3]. Comparison of the amino-acid sequences of carbohydrate intermediates which can be shunted into transaldolases reveals overall sequence identities of greater other metabolic pathways such as glycolysis. The pathway than 50% between prokaryotic and eukaryotic species, for is very flexible and can adjust itself to the various chang- example 53% identity between the enzymes from E. coli ing requirements of the cell, for example, for metabolic and Saccharomyces cerevisiae and 55% identity between intermediates or reducing power in the form of NADPH. E. coli and human transaldolase [3,6,7]. Transaldolase (D-sedoheptulose-7-phosphate:D-glyceralde- Aldolases are of considerable interest as catalysts in hyde-3-phosphate dihydroxyacetone transferase, EC stereospecific organic synthesis. In particular, fructose- 2.2.1.2), one of the enzymes in the non-oxidative branch of 1,6-phosphate aldolase (F-1,6-P aldolase) has been suc- the pentose phosphate pathway, catalyzes the reversible cessfully employed for stereospecific carbon–carbon bond transfer of a dihydroxyacetone moiety, derived from formation in the synthesis of monosaccharides [8]. As part 716 Structure 1996, Vol 4 No 6 Figure 1 Reactions catalyzed by (a) transaldolase and (a) transaldolase (b) CH2OH fructose-1,6-bisphosphate aldolase. D-erythrose CH OH CO 2 H O 4-phosphate H2O H2O CO CH2OH C HOC H ENH2+ HOC H ENC + H COH ENH2 + HOHC 2- HOHC CH2OH CH2OPO3 HOHC HOHC H COH 2- 2- CH2OPO3 CH2OPO3 D-fructose Schiff-base D-glyceraldehyde D-sedoheptulose 6-phosphate intermediate 3-phosphate 7-phosphate (b) fructose-1,6-bisphosphate aldolase CH OPO 2- 2 3 H O H2O 2- H O 2- C O CH2OPO3 C 2 CH2OPO3 ENH2+ HO C H ENC +HCOH ENH2+ CO 2- HOHC CH2OH CH2OPO3 CH2OH HOHC 2- CH2OPO3 D-fructose Schiff-base D-glyceraldehyde Dihydroxyacetone 1,6-bisphophate intermediate 3-phosphate phosphate of on-going efforts to explore the suitability of trans- bind at the same site, Cys239, and the platinum interacts aldolases as catalysts in organic synthesis, we have initi- with the side chain of Met312. The final model consists of ated crystallographic studies of this enzyme [9]. These 2×316 residues and 524 water molecules. One residue, analyses should reveal the molecular architecture of the Asp293, in each subunit was modelled with two alterna- catalyst and provide insights into the structural basis of tive conformations. The protein model has good stereo- the enzymatic mechanism. chemistry: the root mean square (rms) deviation of the bond lengths is 0.006 Å and the rms deviation for In this paper, we describe the three-dimensional (3D) the bond angles is 1.521°. The Ramachandran plot structure of recombinant transaldolase from E. coli at (Fig. 3) shows that 96.5% of the residues are in the most 1.87 Å resolution. We also present the results of a compari- favoured regions of ϕ,f space with no outliers in the son of the structure of transaldolase with structures of disallowed regions. The only residue in the generously other class I aldolases. allowed region (Ser226) has very well defined electron density. The conventional crystallographic R-factor after Results and discussion refinement is 20.1% and R-free is 23.4% in the resolution Structure determination and electron-density map interval 5.5–1.87 Å. The structure of transaldolase was solved by the multiple isomorphous replacement (MIR) method using five heavy Overall structure metal derivatives. After solvent flattening and histogram Structure of the subunit matching, the electron-density map was of sufficient The enzyme subunit consists of a single domain which quality to trace the polypeptide chain of the enzyme. The has the fold of an eight-stranded a/b barrel (Fig. 4). map was subsequently improved by phase combination, Eight parallel b strands (b1–b8) form the core of the using calculated phases from the partial model and the barrel. This core is surrounded by eight a helices MIR phases. In this improved map, the complete biologi- (a1–a8) running approximately antiparallel with the cal amino-acid sequence was fitted into the electron b strands with the exception of helix a8 which has its density. In the final electron-density maps, there is con- helix axis almost perpendicular to strand b8. There are tinuous well-defined electron density for the whole six additional a helices (aA–aF), three of which are polypeptide chain except for a few side chains at the inserted in loop regions between b strands and a helices surface of the molecule. A representative part of the of the barrel, two after strand 2 (aB and aC) and one 2|Fo|–|Fc| electron-density map after completion of the after strand 6 (aD). One additional a helix (aA) is found crystallographic refinement is shown in Figure 2. The at the N terminus and two more helices (aE and aF) overall residue-by-residue real-space correlation [10] be- occur at the C terminus of the polypeptide chain. Two of tween the model and the 2|Fo|–|Fc| electron-density map these helices, aB and aD, point with their N-terminal is 0.89. The heavy metal ions in the mercury derivatives ends into the active site. The helix aF runs across one Research Article Crystal structure of transaldolase Jia et al. 717 Figure 2 Stereodiagram showing part of the final 2Fo–Fc electron-density map for transaldolase, contoured at 1.0s. Figure 3 Structure of the dimer The dimer is formed between one of the subunits in the 180 asymmetric unit and a symmetry mate of the second B ~b 2 b subunit. The dimer interface buries an area of 1600 Å and 135 involves interactions between the N-terminal parts of b ~b helix aF in the two subunits and between the b3–a3 loop ~lSer226 region from one subunit and helix aE from the other. 90 l These interactions involve hydrophobic contacts and hydrogen bonds. 45 L a A One major contribution to this interface area is the inter- Psi (degrees) 0 ~a action between the N-terminal parts of helix aF of the two subunits across a non-crystallographic twofold symme- -45 try axis.
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