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Dextranase from Penicillium minioluteum
Structure, Vol. 11, 1111–1121, September, 2003, 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0969-2126(03)00147-3
Dextranase from Penicillium minioluteum: Reaction Course, Crystal Structure, and Product Complex Anna M. Larsson,1 Rolf Andersson,2 Jerry Sta˚hlberg,3 Lennart Kenne,2 and T. Alwyn Jones1,* 1 Department of Cell and Molecular Biology Biomedical Centre University of Uppsala Box 596 SE-751 24 Uppsala Sweden 2 Department of Chemistry Swedish University of Agriculture Sciences Box 7015 SE-750 07 Uppsala Sweden 3 Department of Molecular Biology Biomedical Centre Swedish University of Agriculture Sciences Box 590 SE-751 24 Uppsala Sweden
Summary Dextranase catalyzes the hydrolysis of the ␣-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme and product-bound forms. The main domain of the enzyme is a righthanded parallel  helix, which is connected to a  sandwich domain at the N terminus. In the structure of the product complex, isomaltose was found to bind in a crevice on the surface of the enzyme. The glycosidic oxygen of the glucose unit in subsite ⫹1 forms a hydrogen bond to the suggested catalytic acid, Asp395. By NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon, implying a single displacement mechanism. Both Asp376 and Asp396 are suitably positioned to activate the water molecule that performs the nucleophilic attack. A new clan that links glycoside hydrolase families 28 and 49 is suggested. Introduction Dextranase (EC 3.2.1.11; ␣-1,6-glucan 6-glucanohydrolase) hydrolyzes the ␣-1,6-glycosidic linkage in dextran polymers. The enzyme cleaves the linkages within the dextran molecule and releases shorter isomaltosaccharides. In the sugar-processing industry, contamination by dextran causes an extensive problem by increasing the viscosity of the sugar juice. The use of dextranase in the processing, however, reduces the viscosity (Clarke, 1997). Dextranase activity has been found in a wide variety of fungi (Kosaric et al., 1973; Koenig and Day, 1988) and bacteria (Dewar and Walker, 1975). Ten dex*Correspondence: [email protected]
tranase genes have been sequenced, but no enzyme structure has been available (Coutinho and Henrissat, 1999), and it is not known whether the reaction mechanism proceeds by inversion or retention of the anomeric carbon. Dextranase enzymes are found in two glycoside hydrolase families (GH), 49 and 66 (Coutinho and Henrissat, 1999), with no sequence similarity between the two families. The bacterial dextranases from the Streptococcus species have been classified into family 66. In family 49, both bacterial dextranases from the Arthrobacter species and fungal dextranases from Penicillium species are found. Dextran 1,6-␣-isomaltotriosidase from Brevibacterium fuscum var. dextranlyticum and isopullulanse from Aspergillus niger belong to the same family 49. It has recently been predicted (Rigden and Franco, 2002) that GH families 49, 55, and 87 share a common evolutionary ancestor with families 28 and 82. P. minioluteum dextranase is a 67 kDa glycoprotein with an optimal activity at pH 5 and an isoelectric point of 3.88 (Raices et al., 1991). The dex gene encoding the enzyme has been cloned and sequenced by Garcia et al. (1996) (GenBank accession number L41562) and expressed in Pichia pastoris at a level of 3.2 g/l (Roca et al., 1996). Since the P. minioluteum dextranase belongs to GH 49, we propose the name Dex49A for the enzyme (Henrissat et al., 1998). To facilitate the crystallographic studies of the protein, we used a glycosylation-free mutant, N5A/N537A/N540A, of Dex49A in this work. In all three potential glycosylation sites, the asparagines were replaced by alanines (Larsson et al., unpublished data). We have previously described the preparation and crystallization of selenomethionine-labeled dextranase (Larsson et al., 2002). In the current publication, we present the three-dimensional structure of the apo-enzyme at 1.8 A˚ resolution and the structure of a product complex at 1.65 A˚ resolution. We also show by NMR that Dex49A is an inverting enzyme, which, together with the structure, allows us to define details of the enzyme mechanism and its relationship to other GH families.
Results and Discussion Structure Determination of Dextranase The structure of a fungal dextranase, Dex49A, from Penicillium miniolutem was determined by the multiple-wavelength anomalous diffraction (MAD) method (Hendrickson, 1991) with selenomethionyl dextranase expressed in the yeast Pichia pastoris. By direct methods, 11 out of 12 selenium sites were located, and the 9 strongest were used for phasing. The native methionyl enzyme had the same space group as, and similar cell parameters to, the selenomethionyl dextranase, but an Riso of 40%. The native structure was therefore solved by molecular replacement with AMoRe (Navaza, 1994) and then refined to 1.8 A˚ resolution. The final model comprises 572 of the 574 amino acids in mature dextranase and 542 water molecules. No interpretable density was present for the
Structure 1112
Table 1. Data Collection Statistics
Synchrotron Wavelength (A˚) Resolution (A˚)a Number of unique reflections Redundancy Program package Completeness (%)a Mean I/Ia Rmerge (%)a,b a
Inflection Point
Peak
Remote
Native
Ligand
X9A; NSLS 0.97922 30.0–2.0 (2.07–2.00) 66,862 3.7 HKL 99.9 (99.6) 18.6 (4.8) 6.2 (23.9)
X9A; NSLS 0.97899 30.0–2.0 (2.07–2.00) 66,907 3.7 HKL 99.8 (99.6) 19.3 (5.1) 6.5 (21.9)
X9A; NSLS 0.96407 30.0–2.0 (2.07–2.00) 67,015 3.7 HKL 99.9 (99.7) 18.0 (4.6) 6.9 (26.3)
ID14 EH1; ESRF 0.934 35.0–1.8 (1.83–1.80) 56,323 7.1 HKL 99.5 (98.8) 26.0 (5.2) 7.5 (40.9)
I711; MAX 1.098 27.5–1.65 (1.74–1.65) 71,784 3.6 CCP4 99.2 (99.2) 7.7 (2.4) 8.2 (37.7)
Values in parentheses refer to the outer resolution shell. Rmerge ⫽ [⌺hkl⌺i|I ⫺ ⬍I⬎|/⌺hkl⌺i|I|] · 100%.
b
two amino acids at the N terminus. The final R factor and Rfree were 14.2% and 16.5%, respectively. Only 3.7% of the nonglycine residues were outside the stringent Ramachandran criteria of Kleywegt and Jones (1996). A complex structure, containing an isomaltose ligand, was also determined and refined. The final refined model had an R factor of 18.8% and an Rfree of 21.5% at 1.65 A˚. Details of the data collection are presented in Table 1, and phasing, refinement, and stereochemistry of the final models are presented in Table 2. Overall Structure of Dextranase Dex49A folds into two domains (Figure 1). The first domain consists of 200 residues forming 13  strands (Figure 2). Nine of those strands, 3–10 and 13, are folded into a  sandwich. The strands in this  sandwich are all antiparallel, with the exception of the interaction between  strands 5 and 13. In the interior of the  sandwich, there are 14 hydrophobic residues that are completely conserved in GH 49. All extended loops are located on the N-terminal side of the  sandwich. This first domain resembles the immunoglobulin fold, but, in
Dex49A, there are two extra  strands, 4 and 5, located beside the C-terminal  strand of the immunoglobulin fold. These two extra strands give the dextranase  sandwich nine  strands, instead of seven, as in immunoglobulins. A Dali (Holm and Sander, 1993) structural similarity search found numerous hits to members of the immunoglobulin superfamily (the top solution had a Z score of 5.2), including a domain in Cel9A from Clostridium thermocellum (Juy et al., 1992; Z score of 2.6). The other domain has a right-handed parallel -helical fold that contains three parallel  sheets. Following the original definition (Yoder et al., 1993b) the three  sheets are named PB1, PB2, and PB3. The  helix contains ten complete coils. In the N-terminal end of the  helix, there are two incomplete coils that only contain the PB2 and PB3  strands, and, in the C terminus, the last coil contains only PB1 and PB2. Hydrophobic and hydrophilic alignment of side chains between different  strands within a  sheet help to stabilize the  helix (Yoder et al., 1993b). One long stretch of stacked residues is present in Dex49A and consists of mostly aliphatic side chains in the interior of the  helix along PB2 (Val268,
Table 2. MAD Phasing and Model Refinement Statistics
Resolution (A˚) Number of sites Mean figure of merit Phasing power Isomorphous RCullis Anomalous RCullis Resolution (A˚) Number of unique reflections Number of reflections in test set Rwork (%) Rfree (%) Number of atoms Protein Ligand Solvent ⬍B⬎ (A˚2) Protein Ligand Solvent Rmsd from ideal valuesa Bond distance (A˚) Bond angle (⬚) a
Ideal values from Engh and Huber (1991).
Peak
Inflection
Remote
2.0 9 0.33 — — 0.87
2.0
2.0
0.20 1.5 0.88
0.66 0.97 0.93
Native
Ligand
24.3–1.80 53417 2850 14.2 16.5
25.7–1.65 68116 3613 18.8 21.5
4391 — 542
4391 23 508
12.8 — 22.0
21.9 25.9 30.5
0.01 1.26
0.01 1.27
Dextranase Structure and Reaction Course 1113
Figure 1. The Structure of Dex49A Successively Colored from Red in the N Terminus to Blue in the C Terminus The right image structure is turned 90⬚ counterclockwise around the vertical axis, relative to the left image.
Ala290, Cys337, Ile362, Val386, Val407, Ile435, Val479, Val505, and Ile538; Figure 3). Two shorter hydrophobic stackings (Ile421 and Ile457; Val482 and Val508) and one short aromatic stacking (Tyr289 and Tyr336) are also present. Asn481 and Asn507 form an asparagine ladder on the outside of T2 of coils 8 and 9 (Figure 3). This ladder is completely conserved in GH 49. The asparagines in the potential glycosylation sites that were replaced with alanines to avoid glycosylation of the protein were Asn5, Asn537, and Asn540. The alanines that replace Asn5 and Asn537 are located at the very N terminus of the  sandwich domain and on the external side of PB2, in the last complete coil of the  helix, respec-
tively. The alanine that replaces Asn540 is sited at an equivalent position to the two asparagines that participate in the asparagine ladder on the coil next to Asn507. None of the three sites are located close to the active site described below. The two domains interact over a large area. The change in buried accessible surface area on forming these interactions is ⵑ4970 A˚2. The  sheet and the loops
Figure 3. The Side Chains of the Hydrophobic Stacking along PB2 in the Interior of the  Helix Are Shown in Gray
Figure 2. Topology Map of the  Sandwich Domain that Comprises the First 200 Amino Acids of the Protein The coloring is derived from Figure 1.
Two asparagines in T2 that form a short asparagine ladder and are conserved within GH 49 are shown in gold to the right. In the interface between the two domains, a large number of amino acids are conserved within the family. These are colored in gold in the upper part of the figure.
Structure 1114
Figure 4. 1H NMR Spectra of Dextran 1H NMR specra of dextran alone (A) and 18 min (B), 1.5 hr (C), and 18 hr after addition of Dex49A.
in the C-terminal end of the  sandwich fold interact with the surface of PB3 and the T3 loops from coils 1 to 9 in the parallel  helix. The interface has 29 amino acids that are completely conserved in GH 49 (Figure 3). The conserved residues participate both in polar and hydrophobic interactions. At the N terminus of the  helix, there is a 14-amino-acid-long linker that connects the two domains. The linker is bound to the surface of PB2, with hydrophobic interactions between Pro208 in the linker and the side chains of Trp267 and Tyr269 on the  helix surface that are conserved within GH 49. The C terminus, on the other hand, has no extension and is covered by a loop between PB3 of the last complete turn and PB1 of the final turn. In addition there are two tryptophans, Trp568 and Trp571, close to the C-terminal end that cap the end of the  helix. These tryptophans are also completely conserved for the dextranases in GH 49. The N terminus of the parallel  helix is not covered by any polypeptide. Dex49A contains six cysteines, four of which form disulphide bridges. Cys9 and Cys14 link a four-residue-
long loop that stabilizes the N terminus of the protein. In the T3 loop of  helix coil 8, the bridge between Cys484 and Cys488 forms a similar loop only three residues long. In the latter case, the disulphide bridge bends the main chain where the two adjacent  helix coils have more-extended T3 loops. One of the two free cysteines, Cys337, is surrounded by aromatic rings in the core of the  helix and participates in the hydrophobic stacking along PB2. The other free cysteine, Cys415, is buried in the interface between the two domains. None of the six cysteines in Dex49A is conserved within GH 49. Predicting the  Helix Fold The BetaWrap computer program (Cowen et al., 2002) scores the sequence for compatibility with the righthanded  helix fold. The  helix fold of Dex49A was recognized by BetaWrap, giving a raw score of ⫺21.29 and a P value of 2.2 · 10⫺3. The P value is a rescaling of the raw score that gives a rough estimate of the likelihood that a randomly chosen non- helix sequence from the Protein Data Bank would give a similar score.
Dextranase Structure and Reaction Course 1115
Figure 5. Isomaltose Is Hydrogen-Bonded (Black Bubbles) to Six Amino Acids in the Protein The density is A-weighted 2Fo ⫺ Fc map (Read, 1986) at 0.4 electrons/A˚3. The O6 hydroxyl group at the nonreducing end of the sugar is hydrogen bonded to the proposed proton donor, Asp395. Two other aspartyl residues, Asp376 and Asp396, are hydrogen bonded to the water molecule that is suggested to perform the nucleophilic attack. The amino acids that are shown in gold are completely conserved within GH 49.
A P value less than 0.01 indicates that it might be a right-handed  helix fold. The other enzymes in GH 49 gave similar P values. BetaWrap did not find any motif for a  helix fold in the Streptococcus dextranases belonging to GH 66. NMR Analysis The enzymatic hydrolysis of dextran with Dex49A was monitored by 1H NMR spectroscopy. The major signals in the spectrum obtained for the polysaccharide (Figure 4) derived from the ␣-1,6-linked glucose residues with the signal for the anomeric proton at ␦ 4.95 ppm, whereas only minor signals were observed for the 1,3 linkages (␦ 5.30 ppm). The enzymatic hydrolysis was followed by 1H NMR spectroscopy, and changes in the spectra were registered. After only a few minutes, the signal for the anomeric proton at ␦ 4.95 ppm had decreased, and new signals at ␦ 4.60 ppm and 3.30 ppm with large coupling constants, well separated from the crowded region, were observed (Figure 4). These signals, identified as signals from H-1 and H-2 of a -D-glucosyl residue at a reducing end (Jansson et al., 1994), increased in intensity during the first part of the reaction. After some time, a signal at ␦ 5.20 ppm from H-1 of a reducing end ␣-D-glucosyl residue occurred and increased in intensity, demonstrating that the somewhat slower mutarotation of the reducing end residue had occurred. The data demonstrate that, in the hydrolysis of the glycosidic bond with Dex49A, the  configuration of the reducing end residue is formed, and, thus, inversion of configuration occurs. The approximately equal intensities of the signals for anomeric protons of the 1,6
Figure 6. Schematic Representation of the Interaction between the Enzyme and the Ligand, Drawn by LIGPLOT (Wallace et al., 1995) The dotted lines are hydrogen bonds or water-mediated hydrogen bonds. The separations are given in A˚ units.
linkage and the reducing end indicated that isomaltose was the major hydrolysis product. The Active Site Crystals of Dex49A that were soaked with the substrate tetra- and pentaisomaltosaccharides gave well-defined electron density, into which we could model isomaltose with the hydroxyl group at reducing end C-1 in the  conformation (Figure 5). Isomaltose is a possible reaction product from short isomaltosaccharide substrates and presumably binds to the ⫹1 and ⫹2 leaving group subsites (Davies et al., 1997). The binding site that we observed is positioned in a depression, formed by the T3 loops of turns 2–8, close to the surface of PB1. The hydroxyl group at C-6 of the nonreducing end sugar forms a hydrogen bond (2.7 A˚) to the Asp395 carboxylate group. This would be the expected position for the catalytic acid involved in the cleavage of an ␣-1,6-glycosidic linkage. One other hydroxyl group and the ring oxygen of the glucosyl unit in subsite ⫹1 interact directly with the protein. The O3 hydroxyl group is hydrogen bonded to the side chain amine group of Lys315 (2.7 A˚), and the ring oxygen interacts with the amide group of Asn417 (3.1 A˚). For the glucosyl unit in subsite ⫹2, only the O3 hydroxyl group forms hydrogen bonds directly to the protein, both to the side chain amine group of Lys447 (2.9 A˚) and the carboxylate group of Glu449 (2.7 A˚). Three water-mediated hydrogen bonds between the protein and the ligand are also present (Figure 6). Asp395 is conserved within GH 49, as are two other aspartyl residues that are positioned in a potential ⫺1 subsite. Asp395 is at a closest distance of 5.1 A˚ to Asp376 and 3.8 A˚ to Asp396. The latter aspartyl residues are hydrogen bonded (2.9 and 2.6 A˚, respectively) to a water molecule, W283, that is suitably positioned for the nucleophilic attack on the anomeric carbon. Either of the aspartyl residues appears to be properly positioned to act as a base in the hydrolytic reaction (Koshland, 1953).
Structure 1116
Figure 7. The Water-Accessible Surface of the Active Site Is Shown in the Image to the Left The isomaltose ligand is partially visible behind Tyr463. The ball and stick image to the right is shown from the same view. A tunnel is created in the active site by the interaction between the side chains of Tyr463 and Asp317. The side chains colored in gold are located in the presumed ⫺1 and ⫺2 subsites and are totally conserved within GH 49.
Trp425 in the T1 loop of coil 7 is located in the vicinity of the putative active site in a position where it could form a good binding site for a glucosyl unit in subsite ⫺1. Tyr463 in the extended T1 loop of coil 8 is situated above Trp425 and forms a tunnel-like binding site for the dextran polymer when hydrogen bonded to Asp317 (Figure 7). Extensive, cellulose binding tunnels are important features of two families of cellobiohydrolase (Divne et al., 1994; Rouvinen et al., 1990), where they act to thread the substrate into and through the active site (Divne et al., 1998; Zou et al., 1999). However, this is much less developed in Dex49A. Indeed, the density for the ring of Tyr463 is not present in the apo-structure. The absence of density for Tyr463 in the apo-structure and high B factors for the residues in its T1 loop indicate a flexibility that may be needed for the dextran polymer to get access to the binding site. When the product is bound to the protein, Tyr463 is more fixed in position, and a clear density can be seen in the ligand bound structure. Trp425 is completely conserved in GH 49, while Tyr463 is substituted by a tryptophan in the Arthrobacter dextranases, but conserved in the rest of GH 49. In the Arthrobacter dextranases Asp317 is replaced by an alanine, which leaves space for the tryptophan to form a similar tunnel. The differences in the T1 loop of coil 8 and a change of position of Trp425 (1.1 A˚) toward Tyr463 are the only significant changes between the native and the ligand-bound structure forms. The residues that we propose to form the ⫺1 and ⫺2 sites are more conserved in GH 49 than the residues in subsites ⫹1 and ⫹2. Trp425, Lys399, Tyr401, Glu379, and Tyr381 (Figure 6) are conserved in the negative subsites, but only Gln374, which packs against the sugar ring in subsite ⫹1, is completely conserved in the positive subsites. All residues forming hydrogen bonds to the isomaltose are conserved between the two Penicillium dextranases.
For the other dextranases, Asn417 is conserved, but substituted by valine in isopullulanase from A. niger. Lys447 and Glu449 are substituted by tryptophan and aspartate, respectively, for B. fuscum and A. globiformis dextranases and asparagine and glycine, respectively, for isopullulanase. Lys315 is substituted by serine for all the non-Penicillium members of the family. Comparison to Other Right-Handed  Helices The first structure solved with a right-handed  helix fold was a pectate lyase (Yoder et al., 1993a). Several other classes of enzymes with the same fold have since been unraveled (Murzin et al., 1995). Among them are pectin lyases (Vitali et al., 1998), which are closely related to pectate lyases, pectin methylesterase (Jenkins et al., 2001), and two families of glycoside hydrolases, galacturonases and carrageenases, belonging to families 28 and 82, respectively. The phage P22 tailspike protein (Steinbacher et al., 1994), a viral adhesion protein that also hydrolyzes glycosidic linkages, forms a trimer, unlike the other right-handed  helices, which are all monomers. A comparison of Dex49A to other similar righthanded  helix structures is shown in Table 3. The  sandwich domain of Dex49A is not present in any of the  helix proteins compared. The P22 tailspike protein has extra domains, but they do not interact with the  helix. Dex49A has the same number of coils as the GH 28  helices and -carrageenase (Michel et al., 2001) in GH 82. Chondroitinase B (Huang et al., 1999) and the P22 tailspike protein have more-extended  helices, each with thirteen complete coils. The amphipathic ␣ helix present in the N-terminal end for the GH 28 and GH 82  helices is not present in Dex49A. The same ␣ helix is present in all the structures in Table 3, except for the low-molecular weight pectate lyase from Bacillus sp. (Akita et al., 2001), which has no extensions at all, and the P.69 pertactin from Bordetella pertussis (Emsley et
Number of Residues
Polygalacturonase 376 (Erwinia carotovora ssp. carotovora) Rhamnogalacturonase A 422 (Aspergillus aculeatus) Endo-Polygalacturonase II 362 (Aspergillus niger) Chondroitinase B 506 (Flavobacterium heparinium) Tailspike protein 542 (Bacteriophage P22) -Carrageenase 464 (Alteromonas fortis) Pectate Lyase E 355 (Erwinia chrysanthemi) Pectin Lyase B 359 (Aspergillus niger) Pectin Methylesterase 342 (Erwinia chrysanthemi) Pectate Lyase C 353 (Erwinia chrysanthemi) Pectate Lyase 197 (Bacillus sp.) P.69 Pertactin 539 (Bordetella pertussis)
Protein (Source) 1.92 2.03 2.11 2.36 2.13 2.12 2.14 2.23 2.31 2.12 2.04 2.20
224 (11.6) 212 (11.3) 205 (7.3) 197 (10.2) 182 (11.5) 151 (9.3) 162 (6.8) 134 (9.7) 140 (8.6) 142 (7.0) 162 (8.6)
RMSD to Dex49A
251 (13.1)
Number of Atoms within a 3.8 A˚ Cutoff (% Sequence Identity)
Table 3. Comparison of Dex49A with Other Known Structures
8.5
9.1
9.3
10.0
9.6
11.4
10.1
13.0
11.5
18.4
14.8
20.7
Dali Z
1DAB
1EE6
1AIR
1QJV
1QCX
1PCL
1H80
1TYV
1DBG
1CZF
1RMG
1BHE
Protein Data Bank Code
Asp392
Asp201
Asp197
Asp223
Equivalent to Asp395
Putative Proton Donor
Glu359
Asp180
Asp177
Asp202
Equivalent to Asp376
Putative Base
Asp395
Asp202
Glu198
Asp224
Equivalent to Asp396
Dextranase Structure and Reaction Course 1117
Structure 1118
al., 1996), which is a large  helix with 16 coils, but without an extension at the N-terminal end. The most similar structures to Dex49A are the galacturonases (Pickersgill et al., 1998; van Santen et al., 1999; Petersen et al., 1997) found in GH 28. The  helices in family 28 are composed of four  sheets. The  strands in the additional  sheet, PB2a, are separated from the N terminus of PB2 by an amino acid in a left-handed ␣ helix conformation. For Dex49A, this criterion is only fulfilled for two coils. Both Gly432 in coil 7 and Asn502 in coil 9 of Dex49A have the ␣L conformation that gives the preceding  strands the characteristics of the  strands in PB2a, but the corresponding amino acid is missing for the adjacent coils. The galacturonases in GH family 28 hydrolyze ␣-1,2- or ␣-1,4-glycosidic linkages with an inverting mechanism (Biely et al., 1996). The inverting glycoside hydrolases have a single displacement reaction, where a base activates a water molecule and an acid donates a proton to the leaving group (Koshland, 1953). The catalytic groups are invariably a pair of aspartyl/glutamyl residues. For inverting GHs that hydrolyse -linked sugars, these side chains are often separated by ⵑ10 A˚ (McCarter and Withers, 1994). In the galacturonases of family 28, however, the distance between the catalytic residues is significantly shorter. When the enzyme cleaves an ␣-glycosidic linkage, the protonation of the glycosidic oxygen and the nucleophilic attack at the anomeric carbon can be from the same side of the polymer chain (Shimizu et al., 2002). The space normally needed to accommodate both the polymer chain and the nucleophilic water between the catalytic residues located at opposite sides of the polymer is then less. The same appears to be true for Dex49A, which hydrolyses an ␣-1,6-glycosidic linkage. Indeed, the architecture of the active site of the endopolygalacturonases and rhamnogalacturonases in GH 28 is very similar to what we observe in Dex49A. The superimposed active sites of polygalacturonase A from Erwinia carotovora (PehA; Pickersgill et al., 1998) and Dex49A are shown in Figure 8 as an example. Beside the three aspartyl residues, Asp376, Asp395, and Asp396 in Dex49A and Asp202, Asp223, and Asp224 in PehA, that are involved as catalytic residues, two other residues in the active site are conserved between the two enzymes, Thr375 and Gly377 in Dex49A, equivalent to Thr201 and Gly203 in PehA. These two residues are positioned on either side of one of the aspartyl residues that is hydrogen bonded to the nucleophilic water. The threonine forms a hydrogen bond to the backbone of the coil adjacent to the two conserved aspartyls, while the glycine allows a bend in the polypeptide chain. The structure of endopolygalacturonases I (EndoPG I) from Stereum purpureum has been solved in a ternary complex with -D-galactofuranuronate and -D-galactopyranuronate in subsites ⫺1 and ⫹1, respectively (Shimizu et al., 2002). The asparagine in EndoPG I that corresponds to Asn200 in PehA participates in the binding of -D-galactopyranuronate in subsite ⫹1. In GH 49 the asparagine has been substituted for glutamine (Gln374 for Dex49A), which is the only conserved residue in the ⫹1 subsite (Figures 7 and 8). The binding of the ligands in EndoPG I is in accordance with the conclusions that we propose from the binding of isomaltose
Figure 8. The Superimposed Active Sites of Dex49A (Gray) and Polygalacturonase A from E. carotovora (Green) Are Shown The only completely conserved residues between the active sites are the aspartyl residues suggested to be involved in the catalysis and two residues in close vicinity. Gln374 in Dex49A, which is completely conserved in GH 49, substitutes Asn200 in the ⫹1 subsite.
to dextranase. From studying the result of mutations in the two putative bases Asp180 and Asp202 in endopolygalacturonase II from Aspergillus niger (Asp376 and Asp396 in Dex49A), it has been proposed (Armand et al., 2000) that Asp180 is the catalytic base. Mutations of Asp180 to alanine, glutamine, or glutamate all gave inactive enzyme, while the mutation of Asp202 to glutamate gave a highly active enzyme. The similarities in structure and in the architecture of the active site for GH 28 and 49 suggest a new GH clan for these two families. Family 82 also has a  helix fold, but we do not consider it as a member of the new clan, since the suggested active site (Michel et al., 2001) does not show any resemblance to the active sites in families 28 and 49. In Figure 9, the  helix domains of Dex49A, PehA, and -carrageenase from GH 49, 28, and 82, respectively, are shown with the active sites in red. The loops around the active site differ between the dextranase and PehA, but the catalytic residues have equivalent positions. The active site of -carrageenase, on the other hand, has a different location in the  helix, and the catalytic residues are not conserved between GH 82 and the other two families. Biological Implications We present the first crystal structure and reaction course of an enzyme in glycoside hydrolase family 49 and reveal the relation between the enzymes in families 49 and 28. Dextranase from the fungus Penicillium minioluteum is shown to cleave the ␣-1,6-glycosidic linkage in dextran polymers with net inversion of anomeric configuration. The enzyme folds in a right-handed parallel  helix with ten complete coils, similar to the fold in
Dextranase Structure and Reaction Course 1119
Figure 9. Superimposed  Helix Domains The  helix domains of dextranase from P. minioluteum (A), polygalacturonase A from E. carotovora (B), and -Carrageenase from A. fortis (C) GH families 49, 28, and 82, respectively, were superimposed, and the water-accessible surfaces of the three  helices are shown from the same view. The active sites are colored in red.
family 28. In addition, a 200-residue  sandwich domain that interacts extensively with the  helix is present in family 49. The catalytic residues and the architecture in their close vicinity are conserved between the two families. The knowledge about the structure and the reaction course provides important information that can be used in the bioengineering of dextranase for more efficient use of the enzyme in the sugar industry.
NMR Experiments The products formed by the enzymatic degradation of dextran were analyzed by NMR spectroscopy. 1H NMR spectra were obtained for D2O solutions at 30⬚C on a Bruker DRX 400 spectrometer. Dextran (7.7 mg) was dissolved in D2O (0.6 ml), the solution was heated to 30⬚C, and a 1H NMR spectrum was acquired. After the experiment, 10 l of a dextranase solution (1 mg enzyme/ml D2O) was added to the sample, and a number of one-dimensional 1H NMR spectra were acquired over 24 hr.
Experimental Procedures
Comparison to Other Proteins The sequence alignment of the GH 49 enzymes was made with ClustalW (Thompson et al., 1994). Dali (Holm and Sander, 1993) was used to find similar structures in the Protein Data Bank (Berman et al., 2000). The coordinates of proteins with Z scores greater than 6.0 were obtained from the Protein Data Bank and compared with the dextranase structure with the lsq commands in O (Jones et al., 1991). After determining that dextranase includes a right-handed parallel  helix fold, the program BetaWrap (Cowen et al., 2002) was tested to determine whether the fold could be recognized. The BetaWrap program was also used to determine whether a  helix fold could be predicted for the bacterial dextranases in GH family 66. All figures, except Figure 4, were prepared with O and rendered with Molray (Harris and Jones, 2001).
Crystallization and Data Collection Native and selenomethionyl dextranase from P. minioluteum were expressed in the methylotrophic yeast P. pastoris, purified, and crystallized as described previously (Larsson et al., 2002). In the clone used for expression, the three potential glycosylation sites had been removed by site-directed mutagenesis (Larsson et al., in preparation). The native-ligand complex was obtained by soaking native crystals for 2 hr in cryoprotectant with an additional 20 mM mixture of tetra- and pentaisomaltosaccharides. The native methionine apo-enzyme data were collected at beamline ID14-1 at the European Synchrotron Radiation Facility in Grenoble, France. The MAD data set was collected at beamline X9A at the National Synchrotron Light Source at Brookhaven National Laboratory. Three data sets were collected around the selenium K edge: the peak at 12.664 keV, the inflection point at 12.661 keV, and the high-energy remote at 12.860 keV. Data for the native-ligand complex were collected at beamline I711 at MAX-lab in Lund, Sweden. All data sets were collected at 100 K. The native data and the three data sets in the MAD collection were reduced and scaled with the HKL package (Otwinowski, 1993), and the native-ligand complex was reduced and scaled with the CCP4 suite (Collaborative Computational Project Number 4, 1994). Data collection statistics are given in Table 1. Structure Determination and Refinement The structure was solved by the multiple-wavelength anomalous diffraction method (Hendrickson, 1991). The selenium sites were determined from the anomalous signal in the peak data set with SnB (Weeks and Miller, 1999). These sites were refined and phases were calculated with MLPHARE (Otwinowski, 1991) with data to 2.1 A˚. Phases were refined by solvent flattening and histogram matching with DM (Cowtan, 1994). A partial model was generated with ARP/wARP (Perrakis et al., 1999), and a more complete structure was built with O (Jones et al., 1991). The model was improved by alternating cycles of refinement with REFMAC5 (Murshudov et al., 1997) and rebuilding with O. Water molecules were added with ARP/wARP, and their behavior was monitored during refinement. Phasing and model refinement statistics are given in Table 2.
Acknowledgments The authors thank the RapiData 2002 course given at Brookhaven National Laboratory and, especially, Drs. Robert Sweet and K.R. Rajashankar for the help with data collection and structure determination. We are also grateful to Dr. Gunnar Berglund for collecting the ligand complex data at beamline I711 in Lund. We also thank Drs. Jose´ Cremata and Bianca Garcı´a at the Center for Genetic Engineering and Biotechnology in Havana, Cuba, for permission to use the P. pastoris strain expressing P. minioluteum dextranase. This work was supported by a Swedish Science Research Council (VR) grant to T.A.J. Received: March 18, 2003 Accepted: April 4, 2003 Published: September 2, 2003 References Akita, M., Suzuki, A., Kobayashi, T., Ito, S., and Yamane, T. (2001). The first structure of pectate lyase belonging to polysaccharide lyase family 3. Acta Crystallogr. D Biol. Crystallogr. 57, 1786–1792. Armand, S., Wagemaker, M.J., Sanchez-Torres, P., Kester, H.C., van
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DOI 10.1016/S0969-2126(03)00147-3
Dextranase from Penicillium minioluteum: Reaction Course, Crystal Structure, and Product Complex Anna M. Larsson,1 Rolf Andersson,2 Jerry Sta˚hlberg,3 Lennart Kenne,2 and T. Alwyn Jones1,* 1 Department of Cell and Molecular Biology Biomedical Centre University of Uppsala Box 596 SE-751 24 Uppsala Sweden 2 Department of Chemistry Swedish University of Agriculture Sciences Box 7015 SE-750 07 Uppsala Sweden 3 Department of Molecular Biology Biomedical Centre Swedish University of Agriculture Sciences Box 590 SE-751 24 Uppsala Sweden
Summary Dextranase catalyzes the hydrolysis of the ␣-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme and product-bound forms. The main domain of the enzyme is a righthanded parallel  helix, which is connected to a  sandwich domain at the N terminus. In the structure of the product complex, isomaltose was found to bind in a crevice on the surface of the enzyme. The glycosidic oxygen of the glucose unit in subsite ⫹1 forms a hydrogen bond to the suggested catalytic acid, Asp395. By NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon, implying a single displacement mechanism. Both Asp376 and Asp396 are suitably positioned to activate the water molecule that performs the nucleophilic attack. A new clan that links glycoside hydrolase families 28 and 49 is suggested. Introduction Dextranase (EC 3.2.1.11; ␣-1,6-glucan 6-glucanohydrolase) hydrolyzes the ␣-1,6-glycosidic linkage in dextran polymers. The enzyme cleaves the linkages within the dextran molecule and releases shorter isomaltosaccharides. In the sugar-processing industry, contamination by dextran causes an extensive problem by increasing the viscosity of the sugar juice. The use of dextranase in the processing, however, reduces the viscosity (Clarke, 1997). Dextranase activity has been found in a wide variety of fungi (Kosaric et al., 1973; Koenig and Day, 1988) and bacteria (Dewar and Walker, 1975). Ten dex*Correspondence: [email protected]
tranase genes have been sequenced, but no enzyme structure has been available (Coutinho and Henrissat, 1999), and it is not known whether the reaction mechanism proceeds by inversion or retention of the anomeric carbon. Dextranase enzymes are found in two glycoside hydrolase families (GH), 49 and 66 (Coutinho and Henrissat, 1999), with no sequence similarity between the two families. The bacterial dextranases from the Streptococcus species have been classified into family 66. In family 49, both bacterial dextranases from the Arthrobacter species and fungal dextranases from Penicillium species are found. Dextran 1,6-␣-isomaltotriosidase from Brevibacterium fuscum var. dextranlyticum and isopullulanse from Aspergillus niger belong to the same family 49. It has recently been predicted (Rigden and Franco, 2002) that GH families 49, 55, and 87 share a common evolutionary ancestor with families 28 and 82. P. minioluteum dextranase is a 67 kDa glycoprotein with an optimal activity at pH 5 and an isoelectric point of 3.88 (Raices et al., 1991). The dex gene encoding the enzyme has been cloned and sequenced by Garcia et al. (1996) (GenBank accession number L41562) and expressed in Pichia pastoris at a level of 3.2 g/l (Roca et al., 1996). Since the P. minioluteum dextranase belongs to GH 49, we propose the name Dex49A for the enzyme (Henrissat et al., 1998). To facilitate the crystallographic studies of the protein, we used a glycosylation-free mutant, N5A/N537A/N540A, of Dex49A in this work. In all three potential glycosylation sites, the asparagines were replaced by alanines (Larsson et al., unpublished data). We have previously described the preparation and crystallization of selenomethionine-labeled dextranase (Larsson et al., 2002). In the current publication, we present the three-dimensional structure of the apo-enzyme at 1.8 A˚ resolution and the structure of a product complex at 1.65 A˚ resolution. We also show by NMR that Dex49A is an inverting enzyme, which, together with the structure, allows us to define details of the enzyme mechanism and its relationship to other GH families.
Results and Discussion Structure Determination of Dextranase The structure of a fungal dextranase, Dex49A, from Penicillium miniolutem was determined by the multiple-wavelength anomalous diffraction (MAD) method (Hendrickson, 1991) with selenomethionyl dextranase expressed in the yeast Pichia pastoris. By direct methods, 11 out of 12 selenium sites were located, and the 9 strongest were used for phasing. The native methionyl enzyme had the same space group as, and similar cell parameters to, the selenomethionyl dextranase, but an Riso of 40%. The native structure was therefore solved by molecular replacement with AMoRe (Navaza, 1994) and then refined to 1.8 A˚ resolution. The final model comprises 572 of the 574 amino acids in mature dextranase and 542 water molecules. No interpretable density was present for the
Structure 1112
Table 1. Data Collection Statistics
Synchrotron Wavelength (A˚) Resolution (A˚)a Number of unique reflections Redundancy Program package Completeness (%)a Mean I/Ia Rmerge (%)a,b a
Inflection Point
Peak
Remote
Native
Ligand
X9A; NSLS 0.97922 30.0–2.0 (2.07–2.00) 66,862 3.7 HKL 99.9 (99.6) 18.6 (4.8) 6.2 (23.9)
X9A; NSLS 0.97899 30.0–2.0 (2.07–2.00) 66,907 3.7 HKL 99.8 (99.6) 19.3 (5.1) 6.5 (21.9)
X9A; NSLS 0.96407 30.0–2.0 (2.07–2.00) 67,015 3.7 HKL 99.9 (99.7) 18.0 (4.6) 6.9 (26.3)
ID14 EH1; ESRF 0.934 35.0–1.8 (1.83–1.80) 56,323 7.1 HKL 99.5 (98.8) 26.0 (5.2) 7.5 (40.9)
I711; MAX 1.098 27.5–1.65 (1.74–1.65) 71,784 3.6 CCP4 99.2 (99.2) 7.7 (2.4) 8.2 (37.7)
Values in parentheses refer to the outer resolution shell. Rmerge ⫽ [⌺hkl⌺i|I ⫺ ⬍I⬎|/⌺hkl⌺i|I|] · 100%.
b
two amino acids at the N terminus. The final R factor and Rfree were 14.2% and 16.5%, respectively. Only 3.7% of the nonglycine residues were outside the stringent Ramachandran criteria of Kleywegt and Jones (1996). A complex structure, containing an isomaltose ligand, was also determined and refined. The final refined model had an R factor of 18.8% and an Rfree of 21.5% at 1.65 A˚. Details of the data collection are presented in Table 1, and phasing, refinement, and stereochemistry of the final models are presented in Table 2. Overall Structure of Dextranase Dex49A folds into two domains (Figure 1). The first domain consists of 200 residues forming 13  strands (Figure 2). Nine of those strands, 3–10 and 13, are folded into a  sandwich. The strands in this  sandwich are all antiparallel, with the exception of the interaction between  strands 5 and 13. In the interior of the  sandwich, there are 14 hydrophobic residues that are completely conserved in GH 49. All extended loops are located on the N-terminal side of the  sandwich. This first domain resembles the immunoglobulin fold, but, in
Dex49A, there are two extra  strands, 4 and 5, located beside the C-terminal  strand of the immunoglobulin fold. These two extra strands give the dextranase  sandwich nine  strands, instead of seven, as in immunoglobulins. A Dali (Holm and Sander, 1993) structural similarity search found numerous hits to members of the immunoglobulin superfamily (the top solution had a Z score of 5.2), including a domain in Cel9A from Clostridium thermocellum (Juy et al., 1992; Z score of 2.6). The other domain has a right-handed parallel -helical fold that contains three parallel  sheets. Following the original definition (Yoder et al., 1993b) the three  sheets are named PB1, PB2, and PB3. The  helix contains ten complete coils. In the N-terminal end of the  helix, there are two incomplete coils that only contain the PB2 and PB3  strands, and, in the C terminus, the last coil contains only PB1 and PB2. Hydrophobic and hydrophilic alignment of side chains between different  strands within a  sheet help to stabilize the  helix (Yoder et al., 1993b). One long stretch of stacked residues is present in Dex49A and consists of mostly aliphatic side chains in the interior of the  helix along PB2 (Val268,
Table 2. MAD Phasing and Model Refinement Statistics
Resolution (A˚) Number of sites Mean figure of merit Phasing power Isomorphous RCullis Anomalous RCullis Resolution (A˚) Number of unique reflections Number of reflections in test set Rwork (%) Rfree (%) Number of atoms Protein Ligand Solvent ⬍B⬎ (A˚2) Protein Ligand Solvent Rmsd from ideal valuesa Bond distance (A˚) Bond angle (⬚) a
Ideal values from Engh and Huber (1991).
Peak
Inflection
Remote
2.0 9 0.33 — — 0.87
2.0
2.0
0.20 1.5 0.88
0.66 0.97 0.93
Native
Ligand
24.3–1.80 53417 2850 14.2 16.5
25.7–1.65 68116 3613 18.8 21.5
4391 — 542
4391 23 508
12.8 — 22.0
21.9 25.9 30.5
0.01 1.26
0.01 1.27
Dextranase Structure and Reaction Course 1113
Figure 1. The Structure of Dex49A Successively Colored from Red in the N Terminus to Blue in the C Terminus The right image structure is turned 90⬚ counterclockwise around the vertical axis, relative to the left image.
Ala290, Cys337, Ile362, Val386, Val407, Ile435, Val479, Val505, and Ile538; Figure 3). Two shorter hydrophobic stackings (Ile421 and Ile457; Val482 and Val508) and one short aromatic stacking (Tyr289 and Tyr336) are also present. Asn481 and Asn507 form an asparagine ladder on the outside of T2 of coils 8 and 9 (Figure 3). This ladder is completely conserved in GH 49. The asparagines in the potential glycosylation sites that were replaced with alanines to avoid glycosylation of the protein were Asn5, Asn537, and Asn540. The alanines that replace Asn5 and Asn537 are located at the very N terminus of the  sandwich domain and on the external side of PB2, in the last complete coil of the  helix, respec-
tively. The alanine that replaces Asn540 is sited at an equivalent position to the two asparagines that participate in the asparagine ladder on the coil next to Asn507. None of the three sites are located close to the active site described below. The two domains interact over a large area. The change in buried accessible surface area on forming these interactions is ⵑ4970 A˚2. The  sheet and the loops
Figure 3. The Side Chains of the Hydrophobic Stacking along PB2 in the Interior of the  Helix Are Shown in Gray
Figure 2. Topology Map of the  Sandwich Domain that Comprises the First 200 Amino Acids of the Protein The coloring is derived from Figure 1.
Two asparagines in T2 that form a short asparagine ladder and are conserved within GH 49 are shown in gold to the right. In the interface between the two domains, a large number of amino acids are conserved within the family. These are colored in gold in the upper part of the figure.
Structure 1114
Figure 4. 1H NMR Spectra of Dextran 1H NMR specra of dextran alone (A) and 18 min (B), 1.5 hr (C), and 18 hr after addition of Dex49A.
in the C-terminal end of the  sandwich fold interact with the surface of PB3 and the T3 loops from coils 1 to 9 in the parallel  helix. The interface has 29 amino acids that are completely conserved in GH 49 (Figure 3). The conserved residues participate both in polar and hydrophobic interactions. At the N terminus of the  helix, there is a 14-amino-acid-long linker that connects the two domains. The linker is bound to the surface of PB2, with hydrophobic interactions between Pro208 in the linker and the side chains of Trp267 and Tyr269 on the  helix surface that are conserved within GH 49. The C terminus, on the other hand, has no extension and is covered by a loop between PB3 of the last complete turn and PB1 of the final turn. In addition there are two tryptophans, Trp568 and Trp571, close to the C-terminal end that cap the end of the  helix. These tryptophans are also completely conserved for the dextranases in GH 49. The N terminus of the parallel  helix is not covered by any polypeptide. Dex49A contains six cysteines, four of which form disulphide bridges. Cys9 and Cys14 link a four-residue-
long loop that stabilizes the N terminus of the protein. In the T3 loop of  helix coil 8, the bridge between Cys484 and Cys488 forms a similar loop only three residues long. In the latter case, the disulphide bridge bends the main chain where the two adjacent  helix coils have more-extended T3 loops. One of the two free cysteines, Cys337, is surrounded by aromatic rings in the core of the  helix and participates in the hydrophobic stacking along PB2. The other free cysteine, Cys415, is buried in the interface between the two domains. None of the six cysteines in Dex49A is conserved within GH 49. Predicting the  Helix Fold The BetaWrap computer program (Cowen et al., 2002) scores the sequence for compatibility with the righthanded  helix fold. The  helix fold of Dex49A was recognized by BetaWrap, giving a raw score of ⫺21.29 and a P value of 2.2 · 10⫺3. The P value is a rescaling of the raw score that gives a rough estimate of the likelihood that a randomly chosen non- helix sequence from the Protein Data Bank would give a similar score.
Dextranase Structure and Reaction Course 1115
Figure 5. Isomaltose Is Hydrogen-Bonded (Black Bubbles) to Six Amino Acids in the Protein The density is A-weighted 2Fo ⫺ Fc map (Read, 1986) at 0.4 electrons/A˚3. The O6 hydroxyl group at the nonreducing end of the sugar is hydrogen bonded to the proposed proton donor, Asp395. Two other aspartyl residues, Asp376 and Asp396, are hydrogen bonded to the water molecule that is suggested to perform the nucleophilic attack. The amino acids that are shown in gold are completely conserved within GH 49.
A P value less than 0.01 indicates that it might be a right-handed  helix fold. The other enzymes in GH 49 gave similar P values. BetaWrap did not find any motif for a  helix fold in the Streptococcus dextranases belonging to GH 66. NMR Analysis The enzymatic hydrolysis of dextran with Dex49A was monitored by 1H NMR spectroscopy. The major signals in the spectrum obtained for the polysaccharide (Figure 4) derived from the ␣-1,6-linked glucose residues with the signal for the anomeric proton at ␦ 4.95 ppm, whereas only minor signals were observed for the 1,3 linkages (␦ 5.30 ppm). The enzymatic hydrolysis was followed by 1H NMR spectroscopy, and changes in the spectra were registered. After only a few minutes, the signal for the anomeric proton at ␦ 4.95 ppm had decreased, and new signals at ␦ 4.60 ppm and 3.30 ppm with large coupling constants, well separated from the crowded region, were observed (Figure 4). These signals, identified as signals from H-1 and H-2 of a -D-glucosyl residue at a reducing end (Jansson et al., 1994), increased in intensity during the first part of the reaction. After some time, a signal at ␦ 5.20 ppm from H-1 of a reducing end ␣-D-glucosyl residue occurred and increased in intensity, demonstrating that the somewhat slower mutarotation of the reducing end residue had occurred. The data demonstrate that, in the hydrolysis of the glycosidic bond with Dex49A, the  configuration of the reducing end residue is formed, and, thus, inversion of configuration occurs. The approximately equal intensities of the signals for anomeric protons of the 1,6
Figure 6. Schematic Representation of the Interaction between the Enzyme and the Ligand, Drawn by LIGPLOT (Wallace et al., 1995) The dotted lines are hydrogen bonds or water-mediated hydrogen bonds. The separations are given in A˚ units.
linkage and the reducing end indicated that isomaltose was the major hydrolysis product. The Active Site Crystals of Dex49A that were soaked with the substrate tetra- and pentaisomaltosaccharides gave well-defined electron density, into which we could model isomaltose with the hydroxyl group at reducing end C-1 in the  conformation (Figure 5). Isomaltose is a possible reaction product from short isomaltosaccharide substrates and presumably binds to the ⫹1 and ⫹2 leaving group subsites (Davies et al., 1997). The binding site that we observed is positioned in a depression, formed by the T3 loops of turns 2–8, close to the surface of PB1. The hydroxyl group at C-6 of the nonreducing end sugar forms a hydrogen bond (2.7 A˚) to the Asp395 carboxylate group. This would be the expected position for the catalytic acid involved in the cleavage of an ␣-1,6-glycosidic linkage. One other hydroxyl group and the ring oxygen of the glucosyl unit in subsite ⫹1 interact directly with the protein. The O3 hydroxyl group is hydrogen bonded to the side chain amine group of Lys315 (2.7 A˚), and the ring oxygen interacts with the amide group of Asn417 (3.1 A˚). For the glucosyl unit in subsite ⫹2, only the O3 hydroxyl group forms hydrogen bonds directly to the protein, both to the side chain amine group of Lys447 (2.9 A˚) and the carboxylate group of Glu449 (2.7 A˚). Three water-mediated hydrogen bonds between the protein and the ligand are also present (Figure 6). Asp395 is conserved within GH 49, as are two other aspartyl residues that are positioned in a potential ⫺1 subsite. Asp395 is at a closest distance of 5.1 A˚ to Asp376 and 3.8 A˚ to Asp396. The latter aspartyl residues are hydrogen bonded (2.9 and 2.6 A˚, respectively) to a water molecule, W283, that is suitably positioned for the nucleophilic attack on the anomeric carbon. Either of the aspartyl residues appears to be properly positioned to act as a base in the hydrolytic reaction (Koshland, 1953).
Structure 1116
Figure 7. The Water-Accessible Surface of the Active Site Is Shown in the Image to the Left The isomaltose ligand is partially visible behind Tyr463. The ball and stick image to the right is shown from the same view. A tunnel is created in the active site by the interaction between the side chains of Tyr463 and Asp317. The side chains colored in gold are located in the presumed ⫺1 and ⫺2 subsites and are totally conserved within GH 49.
Trp425 in the T1 loop of coil 7 is located in the vicinity of the putative active site in a position where it could form a good binding site for a glucosyl unit in subsite ⫺1. Tyr463 in the extended T1 loop of coil 8 is situated above Trp425 and forms a tunnel-like binding site for the dextran polymer when hydrogen bonded to Asp317 (Figure 7). Extensive, cellulose binding tunnels are important features of two families of cellobiohydrolase (Divne et al., 1994; Rouvinen et al., 1990), where they act to thread the substrate into and through the active site (Divne et al., 1998; Zou et al., 1999). However, this is much less developed in Dex49A. Indeed, the density for the ring of Tyr463 is not present in the apo-structure. The absence of density for Tyr463 in the apo-structure and high B factors for the residues in its T1 loop indicate a flexibility that may be needed for the dextran polymer to get access to the binding site. When the product is bound to the protein, Tyr463 is more fixed in position, and a clear density can be seen in the ligand bound structure. Trp425 is completely conserved in GH 49, while Tyr463 is substituted by a tryptophan in the Arthrobacter dextranases, but conserved in the rest of GH 49. In the Arthrobacter dextranases Asp317 is replaced by an alanine, which leaves space for the tryptophan to form a similar tunnel. The differences in the T1 loop of coil 8 and a change of position of Trp425 (1.1 A˚) toward Tyr463 are the only significant changes between the native and the ligand-bound structure forms. The residues that we propose to form the ⫺1 and ⫺2 sites are more conserved in GH 49 than the residues in subsites ⫹1 and ⫹2. Trp425, Lys399, Tyr401, Glu379, and Tyr381 (Figure 6) are conserved in the negative subsites, but only Gln374, which packs against the sugar ring in subsite ⫹1, is completely conserved in the positive subsites. All residues forming hydrogen bonds to the isomaltose are conserved between the two Penicillium dextranases.
For the other dextranases, Asn417 is conserved, but substituted by valine in isopullulanase from A. niger. Lys447 and Glu449 are substituted by tryptophan and aspartate, respectively, for B. fuscum and A. globiformis dextranases and asparagine and glycine, respectively, for isopullulanase. Lys315 is substituted by serine for all the non-Penicillium members of the family. Comparison to Other Right-Handed  Helices The first structure solved with a right-handed  helix fold was a pectate lyase (Yoder et al., 1993a). Several other classes of enzymes with the same fold have since been unraveled (Murzin et al., 1995). Among them are pectin lyases (Vitali et al., 1998), which are closely related to pectate lyases, pectin methylesterase (Jenkins et al., 2001), and two families of glycoside hydrolases, galacturonases and carrageenases, belonging to families 28 and 82, respectively. The phage P22 tailspike protein (Steinbacher et al., 1994), a viral adhesion protein that also hydrolyzes glycosidic linkages, forms a trimer, unlike the other right-handed  helices, which are all monomers. A comparison of Dex49A to other similar righthanded  helix structures is shown in Table 3. The  sandwich domain of Dex49A is not present in any of the  helix proteins compared. The P22 tailspike protein has extra domains, but they do not interact with the  helix. Dex49A has the same number of coils as the GH 28  helices and -carrageenase (Michel et al., 2001) in GH 82. Chondroitinase B (Huang et al., 1999) and the P22 tailspike protein have more-extended  helices, each with thirteen complete coils. The amphipathic ␣ helix present in the N-terminal end for the GH 28 and GH 82  helices is not present in Dex49A. The same ␣ helix is present in all the structures in Table 3, except for the low-molecular weight pectate lyase from Bacillus sp. (Akita et al., 2001), which has no extensions at all, and the P.69 pertactin from Bordetella pertussis (Emsley et
Number of Residues
Polygalacturonase 376 (Erwinia carotovora ssp. carotovora) Rhamnogalacturonase A 422 (Aspergillus aculeatus) Endo-Polygalacturonase II 362 (Aspergillus niger) Chondroitinase B 506 (Flavobacterium heparinium) Tailspike protein 542 (Bacteriophage P22) -Carrageenase 464 (Alteromonas fortis) Pectate Lyase E 355 (Erwinia chrysanthemi) Pectin Lyase B 359 (Aspergillus niger) Pectin Methylesterase 342 (Erwinia chrysanthemi) Pectate Lyase C 353 (Erwinia chrysanthemi) Pectate Lyase 197 (Bacillus sp.) P.69 Pertactin 539 (Bordetella pertussis)
Protein (Source) 1.92 2.03 2.11 2.36 2.13 2.12 2.14 2.23 2.31 2.12 2.04 2.20
224 (11.6) 212 (11.3) 205 (7.3) 197 (10.2) 182 (11.5) 151 (9.3) 162 (6.8) 134 (9.7) 140 (8.6) 142 (7.0) 162 (8.6)
RMSD to Dex49A
251 (13.1)
Number of Atoms within a 3.8 A˚ Cutoff (% Sequence Identity)
Table 3. Comparison of Dex49A with Other Known Structures
8.5
9.1
9.3
10.0
9.6
11.4
10.1
13.0
11.5
18.4
14.8
20.7
Dali Z
1DAB
1EE6
1AIR
1QJV
1QCX
1PCL
1H80
1TYV
1DBG
1CZF
1RMG
1BHE
Protein Data Bank Code
Asp392
Asp201
Asp197
Asp223
Equivalent to Asp395
Putative Proton Donor
Glu359
Asp180
Asp177
Asp202
Equivalent to Asp376
Putative Base
Asp395
Asp202
Glu198
Asp224
Equivalent to Asp396
Dextranase Structure and Reaction Course 1117
Structure 1118
al., 1996), which is a large  helix with 16 coils, but without an extension at the N-terminal end. The most similar structures to Dex49A are the galacturonases (Pickersgill et al., 1998; van Santen et al., 1999; Petersen et al., 1997) found in GH 28. The  helices in family 28 are composed of four  sheets. The  strands in the additional  sheet, PB2a, are separated from the N terminus of PB2 by an amino acid in a left-handed ␣ helix conformation. For Dex49A, this criterion is only fulfilled for two coils. Both Gly432 in coil 7 and Asn502 in coil 9 of Dex49A have the ␣L conformation that gives the preceding  strands the characteristics of the  strands in PB2a, but the corresponding amino acid is missing for the adjacent coils. The galacturonases in GH family 28 hydrolyze ␣-1,2- or ␣-1,4-glycosidic linkages with an inverting mechanism (Biely et al., 1996). The inverting glycoside hydrolases have a single displacement reaction, where a base activates a water molecule and an acid donates a proton to the leaving group (Koshland, 1953). The catalytic groups are invariably a pair of aspartyl/glutamyl residues. For inverting GHs that hydrolyse -linked sugars, these side chains are often separated by ⵑ10 A˚ (McCarter and Withers, 1994). In the galacturonases of family 28, however, the distance between the catalytic residues is significantly shorter. When the enzyme cleaves an ␣-glycosidic linkage, the protonation of the glycosidic oxygen and the nucleophilic attack at the anomeric carbon can be from the same side of the polymer chain (Shimizu et al., 2002). The space normally needed to accommodate both the polymer chain and the nucleophilic water between the catalytic residues located at opposite sides of the polymer is then less. The same appears to be true for Dex49A, which hydrolyses an ␣-1,6-glycosidic linkage. Indeed, the architecture of the active site of the endopolygalacturonases and rhamnogalacturonases in GH 28 is very similar to what we observe in Dex49A. The superimposed active sites of polygalacturonase A from Erwinia carotovora (PehA; Pickersgill et al., 1998) and Dex49A are shown in Figure 8 as an example. Beside the three aspartyl residues, Asp376, Asp395, and Asp396 in Dex49A and Asp202, Asp223, and Asp224 in PehA, that are involved as catalytic residues, two other residues in the active site are conserved between the two enzymes, Thr375 and Gly377 in Dex49A, equivalent to Thr201 and Gly203 in PehA. These two residues are positioned on either side of one of the aspartyl residues that is hydrogen bonded to the nucleophilic water. The threonine forms a hydrogen bond to the backbone of the coil adjacent to the two conserved aspartyls, while the glycine allows a bend in the polypeptide chain. The structure of endopolygalacturonases I (EndoPG I) from Stereum purpureum has been solved in a ternary complex with -D-galactofuranuronate and -D-galactopyranuronate in subsites ⫺1 and ⫹1, respectively (Shimizu et al., 2002). The asparagine in EndoPG I that corresponds to Asn200 in PehA participates in the binding of -D-galactopyranuronate in subsite ⫹1. In GH 49 the asparagine has been substituted for glutamine (Gln374 for Dex49A), which is the only conserved residue in the ⫹1 subsite (Figures 7 and 8). The binding of the ligands in EndoPG I is in accordance with the conclusions that we propose from the binding of isomaltose
Figure 8. The Superimposed Active Sites of Dex49A (Gray) and Polygalacturonase A from E. carotovora (Green) Are Shown The only completely conserved residues between the active sites are the aspartyl residues suggested to be involved in the catalysis and two residues in close vicinity. Gln374 in Dex49A, which is completely conserved in GH 49, substitutes Asn200 in the ⫹1 subsite.
to dextranase. From studying the result of mutations in the two putative bases Asp180 and Asp202 in endopolygalacturonase II from Aspergillus niger (Asp376 and Asp396 in Dex49A), it has been proposed (Armand et al., 2000) that Asp180 is the catalytic base. Mutations of Asp180 to alanine, glutamine, or glutamate all gave inactive enzyme, while the mutation of Asp202 to glutamate gave a highly active enzyme. The similarities in structure and in the architecture of the active site for GH 28 and 49 suggest a new GH clan for these two families. Family 82 also has a  helix fold, but we do not consider it as a member of the new clan, since the suggested active site (Michel et al., 2001) does not show any resemblance to the active sites in families 28 and 49. In Figure 9, the  helix domains of Dex49A, PehA, and -carrageenase from GH 49, 28, and 82, respectively, are shown with the active sites in red. The loops around the active site differ between the dextranase and PehA, but the catalytic residues have equivalent positions. The active site of -carrageenase, on the other hand, has a different location in the  helix, and the catalytic residues are not conserved between GH 82 and the other two families. Biological Implications We present the first crystal structure and reaction course of an enzyme in glycoside hydrolase family 49 and reveal the relation between the enzymes in families 49 and 28. Dextranase from the fungus Penicillium minioluteum is shown to cleave the ␣-1,6-glycosidic linkage in dextran polymers with net inversion of anomeric configuration. The enzyme folds in a right-handed parallel  helix with ten complete coils, similar to the fold in
Dextranase Structure and Reaction Course 1119
Figure 9. Superimposed  Helix Domains The  helix domains of dextranase from P. minioluteum (A), polygalacturonase A from E. carotovora (B), and -Carrageenase from A. fortis (C) GH families 49, 28, and 82, respectively, were superimposed, and the water-accessible surfaces of the three  helices are shown from the same view. The active sites are colored in red.
family 28. In addition, a 200-residue  sandwich domain that interacts extensively with the  helix is present in family 49. The catalytic residues and the architecture in their close vicinity are conserved between the two families. The knowledge about the structure and the reaction course provides important information that can be used in the bioengineering of dextranase for more efficient use of the enzyme in the sugar industry.
NMR Experiments The products formed by the enzymatic degradation of dextran were analyzed by NMR spectroscopy. 1H NMR spectra were obtained for D2O solutions at 30⬚C on a Bruker DRX 400 spectrometer. Dextran (7.7 mg) was dissolved in D2O (0.6 ml), the solution was heated to 30⬚C, and a 1H NMR spectrum was acquired. After the experiment, 10 l of a dextranase solution (1 mg enzyme/ml D2O) was added to the sample, and a number of one-dimensional 1H NMR spectra were acquired over 24 hr.
Experimental Procedures
Comparison to Other Proteins The sequence alignment of the GH 49 enzymes was made with ClustalW (Thompson et al., 1994). Dali (Holm and Sander, 1993) was used to find similar structures in the Protein Data Bank (Berman et al., 2000). The coordinates of proteins with Z scores greater than 6.0 were obtained from the Protein Data Bank and compared with the dextranase structure with the lsq commands in O (Jones et al., 1991). After determining that dextranase includes a right-handed parallel  helix fold, the program BetaWrap (Cowen et al., 2002) was tested to determine whether the fold could be recognized. The BetaWrap program was also used to determine whether a  helix fold could be predicted for the bacterial dextranases in GH family 66. All figures, except Figure 4, were prepared with O and rendered with Molray (Harris and Jones, 2001).
Crystallization and Data Collection Native and selenomethionyl dextranase from P. minioluteum were expressed in the methylotrophic yeast P. pastoris, purified, and crystallized as described previously (Larsson et al., 2002). In the clone used for expression, the three potential glycosylation sites had been removed by site-directed mutagenesis (Larsson et al., in preparation). The native-ligand complex was obtained by soaking native crystals for 2 hr in cryoprotectant with an additional 20 mM mixture of tetra- and pentaisomaltosaccharides. The native methionine apo-enzyme data were collected at beamline ID14-1 at the European Synchrotron Radiation Facility in Grenoble, France. The MAD data set was collected at beamline X9A at the National Synchrotron Light Source at Brookhaven National Laboratory. Three data sets were collected around the selenium K edge: the peak at 12.664 keV, the inflection point at 12.661 keV, and the high-energy remote at 12.860 keV. Data for the native-ligand complex were collected at beamline I711 at MAX-lab in Lund, Sweden. All data sets were collected at 100 K. The native data and the three data sets in the MAD collection were reduced and scaled with the HKL package (Otwinowski, 1993), and the native-ligand complex was reduced and scaled with the CCP4 suite (Collaborative Computational Project Number 4, 1994). Data collection statistics are given in Table 1. Structure Determination and Refinement The structure was solved by the multiple-wavelength anomalous diffraction method (Hendrickson, 1991). The selenium sites were determined from the anomalous signal in the peak data set with SnB (Weeks and Miller, 1999). These sites were refined and phases were calculated with MLPHARE (Otwinowski, 1991) with data to 2.1 A˚. Phases were refined by solvent flattening and histogram matching with DM (Cowtan, 1994). A partial model was generated with ARP/wARP (Perrakis et al., 1999), and a more complete structure was built with O (Jones et al., 1991). The model was improved by alternating cycles of refinement with REFMAC5 (Murshudov et al., 1997) and rebuilding with O. Water molecules were added with ARP/wARP, and their behavior was monitored during refinement. Phasing and model refinement statistics are given in Table 2.
Acknowledgments The authors thank the RapiData 2002 course given at Brookhaven National Laboratory and, especially, Drs. Robert Sweet and K.R. Rajashankar for the help with data collection and structure determination. We are also grateful to Dr. Gunnar Berglund for collecting the ligand complex data at beamline I711 in Lund. We also thank Drs. Jose´ Cremata and Bianca Garcı´a at the Center for Genetic Engineering and Biotechnology in Havana, Cuba, for permission to use the P. pastoris strain expressing P. minioluteum dextranase. This work was supported by a Swedish Science Research Council (VR) grant to T.A.J. Received: March 18, 2003 Accepted: April 4, 2003 Published: September 2, 2003 References Akita, M., Suzuki, A., Kobayashi, T., Ito, S., and Yamane, T. (2001). The first structure of pectate lyase belonging to polysaccharide lyase family 3. Acta Crystallogr. D Biol. Crystallogr. 57, 1786–1792. Armand, S., Wagemaker, M.J., Sanchez-Torres, P., Kester, H.C., van
Structure 1120
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