From the
Institut für Chemie, Takustrasse 6, Freie Universität Berlin, D-14195 Berlin,
Institut für Mikrobiologie und Genetik, Grisebachstrasse 8, Georg-August-Universität, D-37077 Göttingen, Germany,
|| Biotechnologisch-Biomedizinisches Zentrum, Am Deutschen Platz 5, Universität Leipzig, D-04103 Leipzig, Germany
Received for publication, November 14, 2002
, and in revised form, February 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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The general hydrolysis reaction of many glucosidases is well understood, involving either a single-step general acid/base catalysis or a double displacement mechanism, both involving a nucleophile-stabilized, oxocarbenium ion-like transition state and a pair of carboxylic acids in the active site (3). The first mechanism results in an inversion of the stereochemistry around the anomeric carbon, whereas in the second it is retained (4).
Glycoside hydrolase family 4 (GH4)1 is a unique family of glucosidases that requires unusual cofactors and activation conditions, pointing toward a novel type of reaction mechanism (5, 6, 7, 8, 9). All except one of the GH4 enzymes investigated so far have shown a prerequisite for NAD+ and a divalent cation, and some enzymes also require the addition of a reducing agent such as dithiothreitol or -mercaptoethanol for activity (5, 6, 7, 8, 9). The requirement of NAD+ for the hydrolysis reaction of a glycosidic bond has been described so far only for GH4 enzymes.
The role of NAD+ for the activity of these enzymes is not yet understood. A sequence pattern at the N terminus of the GH4 enzymes is related to the "fingerprint" motif found in many of the classical NAD-binding enzymes (6). Mutations in this GXGS motif demonstrated that it is indeed important for NAD+ binding and for the activity of Thermotoga maritima AglA (10). Spectroscopic measurements did not reveal the formation of NADH as a product or long lived intermediate of the catalytic reaction for several GH4 members (6, 10, 11), but a redox reaction during the hydrolysis reaction cannot be excluded.
Enzymes with various reaction specificities are found in this family, including -glucosidases (T. maritima and Thermotoga neapolitana AglA (5)),
-galactosidases (Escherichia coli MelA (13)), 6-phospho-
-glucosidases (Bacillus subtilis GlvA (6), Fusobacterium mortiferum MalH (7), and Klebsiella pneumoniae AglB (14)), 6-phospho-
-glucosidases (E. coli and B. subtilis CelF (11)), and an
-D-glucuronidase (T. maritima Agu (9)). The GH4 family is unique in being the only family so far identified to contain enzymes that are specific for
-glycosidic bonds as well as enzymes that display
-specificity.
Currently no structures are available for a GH4 enzyme. In our ongoing effort to elucidate the catalytic mechanism of these enzymes, we have crystallized the 110-kDa homodimeric glucosidase AglA from T. maritima. Here we present the x-ray structure of the enzyme complexed with NAD+ and the substrate maltose. The 1.9-Å resolution structure permits both a detailed description of the active site arrangement and implications regarding the catalytic mechanism. The structure further defines the evolutionary relationship of this unique enzyme family to other NAD-binding proteins.
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EXPERIMENTAL PROCEDURES |
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Prior to data collection the crystals were transferred stepwise to a buffer containing 20% polyethylene glycol 6000, 1 M LiCl, 25% (v/v) glycerol, and 100 mM Tris/HCl, pH 5.0. After 1030 min of incubation in the cryobuffer, the crystals were frozen in liquid nitrogen or in a nitrogen gas stream at 100 K. Heavy atom derivatives were dissolved in the reservoir buffer and added at a concentration of 5 mM to a 10-µl drop of the mother liquor containing a crystal. This was incubated overnight before being transferred to the cryobuffer and frozen as described.
Data Collection and Data ProcessingX-ray data for four heavy atom derivatives were collected at the BAMline beamline at BESSY II (Berliner Elektronenspeicherring-Gesellschaft für Synchrotron Strahlung mbH II, Berlin, Germany) with a Mar345 imaging plate device as the detector (Table I). Data for two further derivatives and the native data set were collected on a Mar345 imaging plate detector mounted on an Enraf-Nonius FR571 rotating anode source and an Osmic MaxFlux mirror system. The software program Denzo/Scalepack (15) was used for data reduction (Table II).
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Structure Determination and RefinementPhases were determined by the multiple isomorphous replacement and anomalous scattering (MIRAS) method. All crystals belonged to the same crystal form of the space group P21 and were isomorphous with cell dimensions as given in Table II, having two monomers per asymmetric unit and a calculated Matthews parameter of Vm = 2.4. Six heavy atom derivatives have been used for phasing of the native data set to a 1.85-Å resolution. Heavy atom sites and initial phases were determined based on four derivatives collected at the BAMline using the software program SOLVE (16). Additional heavy atom sites of these four derivatives and of the HgCl2 and SmCl3 derivatives were located from difference Fourier maps. Phases were calculated with SHARP (17) using the isomorphous and anomalous contributions. After phase refinement and extension (software program DM (18)), the maps were readily interpretable. A model was built using the program O (19). Programs of the Collaborative Computational Project 4 software suite (18) and the MAPMAN (21), MOLEMAN2 (22), and LSQMAN (23) programs were used for many steps during structure solution and analysis. The structures were refined by simulated annealing and conjugate gradient energy minimization against maximum likelihood targets as implemented in the program CNS (24). Water molecules were picked automatically from Fo Fc electron density maps and verified manually.
Small Angle X-ray ScatteringSAXS measurements were performed on the X33-D1/2 beamline using multiwire proportional chambers with delay line readout at the EMBL outstation in Hamburg on the campus of the Deutsche Elektronen-Synchrotron research center (DESY). The scattering patterns were recorded at a sample detector distance of 1.7 m, equivalent to a range of momentum transfer of 0.02 Å1 < S < 0.35 Å1, with S being the scattering vector. The protein was purified as described (5) and diluted in 0.1 M Tris, pH 7.0, to concentrations between 3 and 15 mg/ml. Ten 1-min exposures at 15 °C were performed on each protein concentration with no protein degradation observed, and a buffer blank measurement was made before and after each protein sample. The programs SAPOKO and PRIMUS2 were used to adjust for beam intensity and detector response, to average the frames, and to subtract the buffer background. Models of the two putative dimers and their fit to the experimental data were evaluated with the program CRYSOL (25).
Structure Analysis and Preparation of FiguresSequence alignment of GH4 enzymes was prepared using MULTALIN (26) and ESPRIPT (27). The topology diagram of AglA was prepared using TOPS (28). Structural homologues were identified using the DALI server (29). All figures of AglA were prepared using a graphical interface3 for MolScript (30), CONSCRIPT (31), and Raster3D (32).
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RESULTS |
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The two AglA molecules in the asymmetric unit that are related by a noncrystallographic 2-fold rotation axis are shown in Fig. 1, a and b. The two molecules are almost identical with the remarkable exception of helix L, which is rotated by 51.7° with respect to the same helix in the other molecule (Fig. 1c). The overall root mean square deviation calculated for C- atoms only between the two monomers is 2.18 Å. This, however, can be dissected into a root mean square deviation of 7.2 Å for residues 316355, including helix L and its encompassing loops, and 0.71 Å for the rest of the structure. This clearly indicates that the majority of the deviation between the two monomers is caused by the different orientation of helix L. Unless noted otherwise, further descriptions of the structure of AglA in this paper concern monomer A.
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Dimer StructureThe dimeric existence of AglA in solution has been confirmed by size exclusion chromatography (5); however, from the crystal structure it is not clear which of the protein contacts represents the true interface. The two possible candidates for the dimeric form of AglA are illustrated in Fig. 1, a and b. Dimer I is formed by hydrogen bond interactions primarily between helices N and O and by hydrophobic interactions between helices I and J. The presence of this dimer in solution would mean that the observed rotation of helix L between the two molecules related by non-crystallographic symmetry is a product of crystal packing, whereas the active sites, which are situated on opposing ends of the dimer, are independent of each other.
The second possible dimer, dimer II (Fig. 1, a and b), is formed by interactions between helices L and M of each subunit. This interface therefore could suggest a physiological role in the dimer formation for the observed rotation of helix L between the monomers related by non-crystallographic symmetry. In this dimer, the two active sites are in close proximity with the loop between helices L and M extending across the cleft of the opposing subunit, thereby reducing the overall surface area of the cleft and the size of the opening to the active site (Fig. 1b). Although smaller than the first interface and involving fewer hydrogen bonds, this second interface comprises a large number of hydrophobic interactions.
Analysis of the buried surface at the respective interfaces showed that for dimer I, 1248 Å2 of the monomeric surface was buried, compared with 1051 Å2 in dimer II. These values are both in the expected range of a native interface (33); however, they are too close to define clearly the true dimer interface. Further investigations were performed using SAXS experiments on the X33-D1/2 EMBL beamline at DESY (Fig. 1d). Differences in the predicted scattering curves for the two possible dimers can be observed in the central region of the curve. The fitted experimental data clearly supports the presence of dimer I in solution with a 2 value reflecting the agreement between the theoretical scattering of the predicted models (
2 = 1 for a perfect fit) and the experimental data of
2 = 0.95 (as opposed to
2 = 1.47 for dimer II). The presence of a 6-Å translation component for the screw operator relating the two monomers of dimer II compared with a 0.2-Å component for dimer I also favors the latter, as protein oligomer subunits are most often related by pure rotation with little or no translational movement (34).
Protein FoldThe overall fold of AglA is shown in Fig. 2, a and b. The monomer has a mixed /
-fold type consisting of 17
-helices (labeled AQ) and 12
-strands (labeled 112). The strands are organized into three
-sheets, which are connected to each other by two hydrogen bonds at each contact. Sheet I consists of
-strands 16 and is a twisted parallel
-sheet, whereas the antiparallel
-sheet II (
-strands 79) shows only a slight twist. Sheet III, also antiparallel, consists of
-strands 1012 and has a severe twist, which causes the sheet to rotate around 130° with reference to its starting point. Interestingly, this exceptional looking morphology is not found in any other glycosidases but is, in fact, a well characterized fold observed in oxidoreductases and sheets II and III, which together with the surrounding
-helices form a typical oxidoreductase fold (CATH code 3.90.110.10
[EC]
(35)). Sheet I is enclosed on both sides by
-helices AG, which are antiparallel to the sheet, and these combine to form a typical NAD-binding Rossman fold consisting of a
-
-
-
-
motif, which extends from residues 4 to 70 and includes the secondary structural elements 1-A-2-B-3. The first segment of this motif (
-
-
) has been predicted previously to extend from residues 4 to 38 in GH4 enzymes (10).
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DISCUSSION |
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A closer investigation of other sugar-converting enzymes containing NAD+-binding folds revealed a similarity to a number of dehydratases, most specifically to the dTDP-D-glucose 4,6-dehydratase (RmlB; Z score, 7.1) from Salmonella enterica (Protein Data Bank code 1KEU [PDB] ) (37). In this dehydratase the NAD+ and 1 sugar ring are bound in positions homologous to those found in AglA. However, the extension of the sugar chain, or in the case of RmlB, the inhibitor, follows different paths. Similarities between the two active sites arise on closer inspection, where, in addition to the well conserved NAD+-binding residues, the coordination of the O-3 and O-4 atoms in the AglA maltose 1 ring by Asn-153 is performed by Tyr-167 in RmlB. The proposed mechanism of RmlB involves an oxidation, dehydration, and reduction step involving NAD+ as the redox cofactor. Also of particular interest is the conservation of AglA Cys-174 as Cys-194 in RmlB. Although participation of Cys-194 in the proposed dehydratase catalytic mechanism has not yet been suggested, the characteristics of AglA Cys-174 imply an importance and role in the active site that will be discussed below.
Active SiteCocrystallization of AglA with NAD+ and maltose clearly identified the location of the active site at the bottom of a deep cleft formed by NAD+ and the loops between -helices A and L,
-sheet 9 and helix I, and the helices G and M (Fig. 3a). The active site is covered by a single long helix (helix L, residues 290316), a dominant feature unique to AglA and not found in related dehydrogenases. The protein without NAD+ therefore contains a tunnel similar in appearance to those of the cellobiohydrolases and
-carrageenases, the only glucosidases characterized previously with such a binding site topology (38, 39). However, although in the cellobiohydrolyases this tunnel has been suggested to aid in feeding long polysaccharides sequentially through the active site (39, 40), in AglA this tunnel is filled effectively by the NAD+ molecule buried deep within it (41). The nicotinamide moiety remains extended into the cavity and thereby creates a deep pocket with the
-nicotinamide ring located at the bottom. The nicotinamide ring is the only part of the NAD+ molecule that does not have a well defined electron density, suggesting that it is flexible in the active site pocket despite the presence of the substrate. The positioning of the nicotinamide moiety of the NAD+ within the active site and in close proximity to the maltose molecule, as well as the biochemical characterization of its requirement for activity, suggest a participation of this group in the catalytic mechanism of AglA.
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Coordination of MaltoseThe electron density clearly indicated the presence of an intact maltose molecule. This demonstrates that the enzyme is inactive in the crystal as maltose is a good substrate and should be hydrolyzed to glucose (5). The nonreducing end of the maltose molecule (1 ring) has a well defined electron density (see Fig. 3a) and is bound by hydrogen bonds to Asn-153, His-203, Asp-260, Asp-119, and Arg-263 and by hydrophobic stacking between Val-117 and the -nicotinamide ring of NAD+ (Fig. 3b). In contrast, the electron density maps indicate that the reducing end (+1 ring) is more flexible, which may be explained by the fact that its only interaction with the protein is a single hydrophobic stacking to Phe-238.
The binding of the substrate molecule by AglA in comparison with the dehydrogenases reveals an interesting relationship. The dehydrogenases contain around the active site three arginine residues, which are responsible for the substrate binding and may exist in an open (Protein Data Bank code 4MDH [PDB] , porcine malate dehydrogenase) or closed state (Protein Data Bank code 1KEU [PDB] , RmlB) (36, 37). Two of these arginines are found on a flexible loop that moves in to cover the binding site when the substrate is present, whereas the third arginine is found in the pocket of the active site (Fig. 3c) (36). The two arginines (Arg-91 and Arg-97) on the mobile loop are conserved in AglA as Arg-97 and Arg-107, which are located on helix D and the following loop, respectively, in positions that correspond to the open conformation of the dehydrogenase. This region is held in the open conformation in the AglA structure by the change from a loop to a helix structure (helix D) and its interaction with the large inserted helix K over the active site. However, another arginine, Arg-263, and an aspartate, Asp-260, occupy the same position as the arginines in the closed form and are supplied from the nonhomologous inserted loop between sheet 9 and helix G. In AglA the third arginine of the dehydrogenase-binding pocket is in the position of the active site disordered loop 6G (between strand 6 and helix G), which consists of residues 175179. Interestingly, these conserved arginines are also present in the dehydratase RmlB, in which they are trapped, as in AglA, in the open conformation away from the active site.
Potential Catalytic Residues of the Active SiteThe active sites of a number of glycosidases now have been characterized structurally, and the catalytic mechanisms have been well described (2, 39). The active site of AglA, however, bears no resemblance to these. We have analyzed the residues surrounding the active site and their sequence conservation in GH4 (Figs. 3b and 4). Instead of an appropriately placed nucleophilic donor on one side of the maltose ring with the corresponding acceptor placed at the required distance to ensure inverting or retaining cleavage, the active site of AglA contains the nicotinamide group of the NAD+ molecule and an array of residues that correspond more to the active sites of dehydrogenases than to those of glucosidases.
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Site-directed mutagenesis on another GH4 enzyme, GlvA from B. subtilis, suggested several residues required for activity (6). GlvA Asp-41 is seen by sequence alignments (Fig. 4) to be equivalent to AglA Asp-39. This residue was predicted to be involved in NAD binding (6), and the AglA structure confirms its position at the end of -sheet 2, interacting with the O-2' and O-3' atoms of the adenine sugar ring. The experiments on GlvA also suggested the residues Glu-111 and Glu-359 as the potential catalytic acid and nucleophile base in the active site. By sequence alignment, these are equivalent to AglA residues Glu-113 and Glu-391, respectively, neither of which is located at the catalytic site. Glu-113 is located at the dimer interface, suggesting that interference with dimerization played a role in the observed loss of activity of the mutant. This is of particular interest as GlvA is shown to be an active homotetramer that requires Mn2+ to tetramerize from homodimers (6). Glu-391 is closer to the active site, situated on the highly twisted
-strand 11 in sheet III. Although not directly involved in the active site, it interacts with Cys-174 via both a water molecule and Asn-202, and through the latter, it also interacts with His-203. Cys-174, Asn-202, and His-203 are conserved completely in GH4, and their roles will be discussed further. We suggest that an involvement of Glu-391 in the positioning of the active site residues explains its requirement for activity.
A single putative nucleophilic donor/acceptor, Asp-260, is located 3.4 Å from the C-1 atom of the maltose. This residue, however, is not conserved in GH4 and lacks the required partner residue on the other side of the maltose molecule. The only totally conserved residues interacting directly with the substrate are His-203 (interacting with O-2 and O-3) and Asn-153 (interacting with O-3 and O-4), located at the bottom of the binding pocket and capable of binding any sugar substrate.
The Cysteine Sulfinic Acid Cys-174 Another completely conserved residue located near the active site is Cys-174. Difference density around the sulfur of this cysteine displayed a "rabbit ear" morphology, which suggested that it was oxidized to a cysteine sulfinic acid (Cys-SO2H) (Fig. 3d). This finding correlates well with the observed requirement for reducing conditions for activity characterized for the protein in vitro, suggesting that this cysteine may be a catalytically important residue in which oxidation causes enzyme inactivation during purification or even during heterologous expression in E. coli. The only other cysteine, Cys-25, is found 24 Å away from the active site and does not show oxidation.
Cysteine sulfinic acids have been described already in several protein structures, primarily as further oxidation states of the catalytically active but unstable cysteine sulfenic acids (Cys-SOHs). Several different roles have been proscribed for these Cys-SOHs. In NADH peroxidase, NADH oxidase, and peroxiredoxins, they function as either catalytically essential redox centers or transient intermediates during peroxide reduction and are thought to be stabilized by a nearby histidine and a potential Mg2+ ion (42). In nitrile hydratases a Cys-SH, Cys-SOH, and Cys-SO2H form a "claw"-like structure that functions to coordinate a Fe(III) ion but plays no catalytic role (43). It is possible in AglA that a Cys-SOH is present in the active protein and is oxidized to Cys-SO2H prior to crystallization.
A functional Cys-SO2H in a glucosidase has been described with the creation of a Glu Cys mutant of the glucoamylase from Aspergillus awamori (44). The accidental oxidation of the catalytic residue mutant led to a 1.6-fold increase in activity compared with the wild type. This is because the more strongly deprotonated Cys-SO2H stabilizes the oxocarbenium ion-like transition state better than a glutamate, hence increasing the kcat (12). Although an active Cys-SO2H has yet to be identified in a wild-type glucosidase, its significance in light of the structural uniqueness of the active site of AglA cannot be ignored.
An opposing theory is the possibility of a role in the inactivation of the enzyme as an artifact of the purification procedure. Cys-SO2H at position 174 is hydrogen-bonded via one of its oxygens to the conserved His-203, a residue which otherwise could interact with the potential nucleophile Asp-260. This could explain in two ways the inactivation of the enzyme: the Cys-SO2H-bound histidine is (a) unavailable to coordinate the Mn2+ ion directly and/or (b) cannot pull Asp-260 into a position closer to the C-1 of the maltose. Either scenario results in the reduced activity, recoverable by reduction, which is observed experimentally. Further experiments are required to determine whether it is this putative role in inactivation or a more direct role that Cys-SO2H at position 174 plays.
Mn2+ BindingDespite being present at concentrations of 10 mM in the crystallization solution, Mn2+ was not located in the structure. According to the metalloprotein data base (20), some of the most common residues involved in coordinating Mn2+ ions in protein structures are histidines, and the binding pocket of AglA has several located nearby the bound maltose. In particular His-175 and His-177 in the disordered loop 6G (B-factor of 52.9 Å2) are situated adjacent to the maltose molecule. At the low pH of the crystallization buffer (pH 4.6), the histidines most probably are protonated and therefore incapable of coordinating the Mn2+. Another putative impediment to Mn2+ coordination is the oxidation of Cys-174 as a coordination ligand, as discussed earlier.
ConclusionThe first crystal structure of a family 4 glucosidase shows that this family represents a new structural clan for the glycoside hydrolases with high structural homology to dehydrogenases. The structure defines the maltose binding mode and shows clearly the binding site of the NAD+ cofactor as a typical Rossman fold. In agreement with previous evidence that NAD+ is absolutely required for activity by these enzymes, the maltose molecule is located in close proximity to NAD+. The finding that the maltose molecule is not turned over in the presence of NAD+, Mn2+, and reducing agents demonstrates that the enzyme as seen in the crystal structure is not in a catalytically active state. Also, a Mn2+ ion could not be located in the density maps. The oxidation of the potential catalytic residue Cys-174 to a sulfinic acid may be the cause of the inactivation; however, a catalytic role for the sulfinic acid is also possible.
Aside from NAD+ and Cys-174, the structure shows additional residues that may play a role in the catalytic mechanism. However, in the absence of additional data on possible catalytic intermediates and the localization of the metal ion, the enzyme mechanism currently remains unclear. In particular, the role of NAD+ has yet to be established, although currently a role in substrate binding, in transition state stabilization via its positive charge, as an acid/base catalyst, or even as a reduction/oxidation mechanism appears possible. These interesting features of the active site, combined with the uniqueness of the structure of AglA compared with other glucosidases and its high homology to dehydrogenases, suggest an exciting new turn in classical views on glycosidase mechanisms.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft. 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 may be addressed. Tel.: 49-551-393795; Fax: 49-551-394897; E-mail: wliebl{at}gwdg.de. To whom correspondence may be addressed. Tel.: 49-30-838-53456; Fax: 49-30-838-56702; E-mail: strater{at}chemie.fu-berlin.de.
1 The abbreviations used are: GH4, glycoside hydrolase family 4; MIRAS, multiple isomorphous replacement and anomalous scattering; SAXS, small angle x-ray scattering.
2 P. V. Konarev, M. H. J. Koch, and D. I. Svergun, manuscript in preparation.
3 N. Sträter, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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