From the
Department of Biological Sciences and Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027,
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey 08854
Received for publication, February 6, 2003
, and in revised form, March 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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In this article, we report the crystal structure of the YdiB protein, which is one of the two shikimate 5-dehydrogenase orthologues in E. coli (12). It is common to find multiple orthologues of individual enzymes in the shikimate pathway in microorganisms. For instance, the genome of Bacillus subtilis (13) carries two versions of the enzyme 3-dehydroquinate dehydratase (products of the aroD and aroQ genes) but just a single version of each of the next two enzymes in the pathway, shikimate 5-dehydrogenase (aroE) and shikimate kinase (aroK). In contrast, this pattern is reversed in the genome of Escherichia coli K12 (14), which carries just a single orthologue of 3-dehydroquinate dehydratase (aroD) but duplicate orthologues for both shikimate 5-dehydrogenase (products of the ydiB and aroE genes) and shikimate kinase (products of the aroK and aroL genes). The ydiB gene in E. coli K12 is cocistronic with the aroD gene, i.e. the only orthologue of 3-dehydroquinate dehydratase found in this organism. The YdiB protein is 28% identical and 50% similar to the AroE protein (12), the other shikimate 5-dehydrogenase orthologue in E. coli, but the two proteins share dramatically stronger sequence conservation in their substrate binding sites (Figs. 2C and 4C).
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Interestingly, the genome of the pathogenic E. coli strain O157 (15) contains one additional orthologue of shikimate 5-dehydrogenase (i.e. three total) and at least two orthologues for every other enzyme in the shikimate pathway. Although the redundancy of these enzymes in many pathogenic organisms suggests that their function is important under physiological conditions, it also raises questions as to the feasibility of using shikimate pathway inhibitors as antibiotics. However, the results reported in this article show very tight conservation of the substrate binding site in all orthologues of shikimate 5-dehydrogenase, suggesting that the design of broad spectrum antibiotics directed against this enzyme is possible (16, 17, 18, 19, 20, 21).
Shikimate 5-dehydrogenase (EC 1.1.1.25 [EC] ) functions to catalyze the reduction of 3-dehydroshikimate to shikimate using the cofactor NADH (12, 14) (Fig. 1). This enzyme belongs to the superfamily of NAD(P)H-dependent oxidoreductases, which function in anabolic and catabolic enzyme pathways as well as in xenobiotic detoxification. This superfamily is usually subdivided into several families, including short chain dehydrogenases (22, 23), medium chain dehydrogenases (24), aldo-keto reductases (25), and iron-activated alcohol dehydrogenases and long chain dehydrogenases (26, 27). The reaction mechanism used by these enzymes involves the catalysis of hydride transfer from the NAD(P)H cofactor on the basis of stabilization of the negative charge accumulation in the hydride-accepting substrate by either an amino acid side chain acting as a general base proton donor or a prosthetic cation in the active site. The crystal structure of the YdiB shikimate dehydrogenase orthologue from E. coli that is reported in this article shows variations in the identity and spatial distribution of the active site residues compared with any other known oxidoreductase structure, suggesting that the details of its catalytic reaction mechanism are likely to be different from the other enzymes in the NAD(P)H-dependent oxidoreductase superfamily.
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MATERIALS AND METHODS |
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Enzyme AssaysActivity was monitored on the basis of the increase of A340 nm on the reduction of NAD+ (assuming an extinction coefficient of 6230 cm-1M-1 for NADH) in the buffer used previously for AroE (30) (100 mm Na2CO3 and 2 mM NAD+, pH 10.6, at 25 °C). The enzyme was used at 320 nM and shikimate or quinate (Sigma) at 4 mM. One unit is defined as a rate of 1 µmol/min.
CrystallizationCrystals were grown using hanging drop vapor diffusion at 21 °C over a reservoir containing 25% PEG 6000, 200 mM ammonium acetate, and 100 mM trisodium citrate dihydrate, pH 5.2. Each drop contained 2 µl of protein, 1.6 µl of reservoir solution, and 0.4 µlof3 M non-detergent sulfobetaine 195. Hexagonal rods grew to 50 x 50 x 400 µm overnight and were frozen in liquid propane using paratone-N as a cryoprotectant. Quinate was added to the protein solution at a 1 mM concentration before setting up the drop that produced the crystal that was solved and refined. However, this ligand was not required for crystal growth, and no evidence of it was observed in the electron density maps.
X-ray Data Collection and Structure DeterminationData were collected from a single crystal at 100 K on beamline X12C of the National Synchrotron Light Source at Brookhaven National Laboratory (Table I). Multiwavelength selenium anomalous diffraction data sets were collected on the Brandeis B4 detector in consecutive 370° sweeps at 0.9790 (peak), 0.9787 (edge), and 0.9200 Å (remote) using 1° oscillations and 510 s exposures. The data were processed and reduced with DENZO and SCALEPACK (31) (Table I). The Laue symmetry of the diffraction pattern and systematic extinctions were consistent with the hexagonal space groups P62 or P64, indicating a 56% solvent content assuming a dimer of YdiB in the asymmetric unit. SOLVE (32) identified 18 of the 22 selenium sites, yielding a map that was used for non-crystallographic symmetry averaging, solvent flattening, and automated model building in RESOLVE (33).
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Model Building and RefinementAlthough only 64% of the backbone and 59% of the side chains were identified during autotracing, the RESOLVE map enabled 81% of the model to be built by hand using O (34). Completion of the structure required iterative cycles of refinement in Crystallography & NMR System (CNS) (35) and manual rebuilding. Eventually, all of the residues in the protomer were included in the model except for the C-terminal affinity tag (and one adjacent residue from the native protein in one of the two subunits). An Rfree set containing 5% of the reflections was selected at random. Strong non-crystallographic symmetry restraints (250 kcal/Å2 and B = 1.5) were maintained throughout the model at all stages of refinement, which consisted of iterations of overall anisotropic B-factor refinement, bulk-solvent correction, rigid-body refinement, positional minimization, individual isotropic B-factor refinement, torsional angle dynamics using slow cooling annealing, and automatic water addition. The final model is described in Table I.
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RESULTS AND DISCUSSION |
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Structure of the YdiB ProtomerThe YdiB protomer is composed of two /
domains plus a pair of C-terminal
-helices that bridge these two domains (Fig. 2). A deep cavity is found at the interface between these domains. This cavity is likely to form the active site of the enzyme on the basis of the fact that it is contiguous with the hydride acceptor site on the NAD+ cofactor, which was found bound to the C-terminal domain in the structure. The apparently flat geometry of the nicotinamide ring suggests that the cofactor is bound in the oxidized NAD+ state (36), although this conclusion is tentative given the resolution of our crystal structure. The cofactor was not added to the crystallization reaction, indicating that it co-purified with the enzyme through two columns and must therefore dissociate very slowly in the absence of substrate.
The N-terminal /
domain of YdiB comprises residues 1105. The mostly parallel six-stranded
-sheet at the core of this domain is flanked on each side by two
-helices, one of them coming from the extreme C terminus of the polypeptide (Fig. 2A). The central
-sheet in this domain has its strands in the order of 2-1-3-5-6-4, with only strand 5 antiparallel to the others (Fig. 2B). A systematic analysis using the DALI program identifies a number of protein domains with significant structural similarity, the closest of which is the N-terminal domain of E. coli IIB cellobiose-specific phosphotransferase (Protein Data Bank number 1iib
[PDB]
) (37), which gives a Z-score of 5.6 for alignment of 72 residues with a root mean square deviation of 2.5 Å. However, neither this domain nor any of the other structural homologues has the same topology as the N-terminal domain in YdiB, suggesting that it is unique among proteins of known structure.
The C-terminal /
domain of YdiB comprises residues 106254, which form a characteristic dinucleotide-binding Rossmann fold (38). The entirely parallel six-stranded
-sheet at the core of this domain is flanked by three
-helices on one side and two on the other (Fig. 2B). The central
-sheet in this domain has its strands in the order of 6-5-4-1-2-3 and only deviates from the canonical Rossmann fold by having one of its flanking
-helices replaced by a long well ordered loop that contains a small 310-helix (between strands
10 and
11 in Fig. 2B). The NAD+ cofactor binds to this domain with its nicotinamide ring in the pro-R conformation (Fig. 2A), indicating that this enzyme is a class A NAD(P)-dependent oxidoreductase like the well studied malate, lactate, and alcohol dehydrogenases.
Structure of the YdiB DimerGel filtration and static lightscattering data indicate that YdiB forms a homodimer in solution (not shown), likely corresponding to the dimer found in the asymmetric unit in the crystal structure. This species buries 2718 Å2 of solvent-accessible surface area in its interface, which is mediated primarily by residues in strand 1 and helices
1 and
2 in the N-terminal
/
domain. Superimposition of the individual domains in the two independent protomers yields root mean square deviations of 0.8 Å for the N-terminal
/
domain, 0.2 Å for the C-terminal
/
domain (i.e. the Rossmann fold), and 0.4 Å for the C-terminal
-helices. Superimposition of just the Rossmann folds (Fig. 3A) shows that a rigid body rotation occurs between the
/
domains when the two protomers in the asymmetric unit are compared. Residue Gly-104 near the C terminus of the N-terminal
/
domain appears to act as a hinge for this rotation, which modulates the width of the active site cavity (Fig. 3A).
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Structural Similarity to the Methylene Tetrahydromethanopterin Dehydrogenase, MtdAAmong proteins of known structure, the MtdA from Methylobacterium extorquens (39) shows the highest sequence homology and structural similarity to the C-terminal Rossmann domain in YdiB (Z-score of 13.7 for alignment of 135/148 residues with a root mean square deviation of 3.0 Å and 27% sequence identity). The structure similarity between the YdiB and MtdA structures extends beyond the boundary of this domain (Fig. 3B). In both enzymes, the active site is at the interface of the two /
domains found in the protomer. Moreover, there is significant structural homology (40) in the N-terminal
/
domains (Z-score of 5.2 for alignment of 71/106 residues with a root mean square deviation of 2.5 Å and 7% sequence identity), despite the failure of iterative position-specific iterated basic alignment search tool analysis (41) to find sequence homology in this region. Although these domains share common topology in their three N-terminal
-strands, their overall topology is different (Figs. 2C and 3B), as observed with the other structural homologues of this domain of YdiB. Furthermore, the putative active site residues differ in MtdA and YdiB (Fig. 2C), indicating likely divergence in enzymatic mechanism.
Although the structurally conserved N-terminal region of the N-terminal /
domain mediates formation of the oligomer interface in both YdiB and MtdA, the structural interactions at the interfaces are very different. Its position has migrated in MtdA relative to YdiB to enable the subunit-subunit interface in this trimeric enzyme to provide an extended recognition site for the much larger substrate that it oxidizes (39) (Fig. 3B). These proteins therefore demonstrate an interesting paradigm whereby structural evolution of an oligomeric interface is used to modify substrate specificity in the NAD(P)H-dependent oxidoreductase superfamily.
Binding Environment of the NAD+ CofactorThe average B-factor of the bound NAD+ molecule is 35.6 Å2 compared with 34.4 Å2 for the overall polypeptide chain. This result suggests that there is full occupancy of the active site by the co-purified NAD+ and reinforces the conclusion that it is bound to the enzyme very tightly. The extended NAD+ molecule makes multiple interactions with the protein (Fig. 4, A and B). The side chain of invariant residue Arg-156 interacts with the adenine, whereas Asn-155 and Asp-158 hydrogen bond (H-bond) to the hydroxyl groups of the adjacent adenosyl ribose. Residues Asp-158 and Glu-159 in the vicinity of the O2' hydroxyl of this ribose explain the specificity of YdiB for binding NAD(H) over NADP(H) on the basis of their ability to mediate electrostatic repulsion of the extra phosphate bound to the adenosyl ribose in NADP(H). Significant heterogeneity is observed in the identity of these residues in other shikimate 5-dehydrogenase orthologues (Fig. 2C), explaining their variable preferences for NAD(H) versus NADP(H). For example, the NADP(H)-dependent shikimate dehydrogenase AroE (12) has valine and serine at positions 158159 and arginine at position 160 instead of the phenylalanine at this position in YdiB. Positively charged residues near this region of the cofactor are strongly correlated with specificity for NADP(H) over NAD(H) on the basis of the ability to form an ionic interaction with the extra phosphate group on the adenosyl ribose (42).
The glycine-rich loop, which anchors the pyrophosphate of the NAD(H) in the active site, has a variant sequence of GXGG in YdiB (Fig. 4, A and B), compared with the GXGXXG "finger-print" motif found in most NAD(P)-dependent oxidoreductases (43). The backbone of this segment forms an N-terminal helix-capping structure in YdiB, which shares a conserved conformation with the canonical motif found in the other superfamily members. As in many of these enzymes (44), the direct H-bonds between the amides of the glycines in the motif and the phosphate oxygens are supplemented by an indirect H-bond via a strongly ordered (B-factor 26 Å2) buried water molecule located in the center of the helix-capping structure. However, YdiB shows an unusual "clamp" over the pyrophosphate and adenosyl ribose formed by residues Lys-205 and Phe-160, respectively, which buries the NAD+ more than in other superfamily members. This clamp may contribute to the very slow release rate of the cofactor from YdiB.
The amide group of the nicotinamide makes extensive contacts with the N-terminal helix-capping motif on 9, including an H-bond to the carbonyl of Gly-255, that hold the ring in the pro-R conformation (Fig. 4, A and B). However, the six atoms of the nicotinamide ring make a total of only three or four van der Waals contacts with the protein. One of these is between the hydride-transferring atom C4N and the carboxylate group of the invariant residue Asp-107. Interestingly, the ring is positioned directly over two side chains containing sulfur atoms, Cys-232 and Met-258. The low pKa of the cysteine residue raises the possibility that it could be deprotonated under some circumstances in the presence of NAD+ to make an ionic interaction with the bound cofactor.
Shikimate Binding Site and Hypothesized Catalytic MechanismAnalyses of the active site of YdiB combined with manual docking exercises suggest that the binding site for the shikimate substrate is located in the cleft between the two /
domains close to the solvent-exposed Re side of the nicotinamide ring (Figs. 3B and 4C). This cavity is lined by a set of phylogenetically conserved residues (e.g. Lys-71, Asn-92, Thr-106, and Asp-107) with chemical characteristics similar to those that mediate the binding of shikimate derivatives in several other enzymes (i.e. 2,3-dehydroquinic acid to type II dehydroquinase (45) and shikimate-3-phosphate to 5-enolpyru-vylshikimate-3-phosphate synthase (46)). Shikimate could be modeled into this site with minimal ambiguity on the basis of the previously established stereochemistry of ternary complexes in other NAD(P)-dependent oxidoreductases (47). Specifically, the carbon containing the hydride donor and the two flanking carbons on shikimate were aligned with the equivalent atoms in the substrate in a crystallographically observed complex, with the ring oriented so that the reactive hydride points toward the C4N acceptor on the nicotinamide ring. This exercise yields a model for the ternary complex (Fig. 4C) in which all but one of the oxygen atoms on the shikimate interact with side chain heteroatoms that are invariant in all shikimate dehydrogenase orthologues (Fig. 2C). Specifically, the C1 carboxylate accepts a bifurcated H-bond from Ser-67, the C4 hydroxyl donates an H-bond to Gln-262, and the C5 hydroxyl donates a bifurcated H-bond to Asp-107. The remaining oxygen atom on shikimate (the C3 hydroxyl) H-bonds with Tyr-234, which is conserved in AroE and 90% of the orthologous sequences (Fig. 2C). Furthermore, invariant residue Lys-71 is close to the carboxylates of both the shikimate and Asp-107. Although these groups are not within H-bonding distance when shikimate is modeled into our crystal structure of the binary complex, a rotamer change and/or the demonstrated rotational flexibility of the N-terminal
/
domain (Fig. 3A) could allow closer interactions to occur in the activated ternary complex.
This model for the ternary complex with shikimate suggests a hypothesis for the catalytic reaction mechanism of YdiB. The presence of an H-bond between Asp-107 and the C5 hydroxyl of the shikimate suggests that the carboxylate side chain will act as the acceptor for the proton from this hydroxyl when the C5 hydride is transferred to NAD+ (right to left in Fig. 1). This mechanism is highly attractive, because the pKa of the aspartate makes it an excellent proton acceptor. When the reaction is run in reverse (left to right in Fig. 1), the principle of microscopic reversibility would then require the aspartate to be protonated before binding 3-dehydroshikimate, which could be promoted by the negative charge on the adjacent NADH. Comparison with all known three-dimensional structures using DALI (40) and motif searches using SPASM (48) shows that the side chain disposition in the active site YdiB is unique, suggesting that it uses a novel catalytic reaction mechanism relative to other enzymes in the NAD(P)-dependent oxidoreductase superfamily. Moreover, the key residues in YdiB that interact with the substrate and cofactor are exceedingly well conserved in all shikimate 5-dehydrogenase orthologues (12, 16, 30) including the AroE protein (12, 30), implying that they all use a similar catalytic mechanism and that the design of broad spectrum inhibitors of this enzyme may be possible.
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FOOTNOTES |
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* This work was supported by Grant NIGMS-P50-GM62413 from the Protein Structure Initiative of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 212-854-5443; Fax: 212-865-8246; E-mail: hunt{at}sid.bio.columbia.edu.
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ACKNOWLEDGMENTS |
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REFERENCES |
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