Crystal structure and snapshots along the reaction pathway of a family 51 -L-arabinofuranosidase
2003; Springer Nature; Volume: 22; Issue: 19 Linguagem: Inglês
10.1093/emboj/cdg494
ISSN1460-2075
Autores Tópico(s)Enzyme Structure and Function
ResumoArticle1 October 2003free access Crystal structure and snapshots along the reaction pathway of a family 51 α-L-arabinofuranosidase Klaus Hövel Klaus Hövel Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Dalia Shallom Dalia Shallom Department of Food Engineering and Biotechnology, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Karsten Niefind Karsten Niefind Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Valery Belakhov Valery Belakhov Department of Chemistry, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Gil Shoham Gil Shoham Department of Inorganic Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel Search for more papers by this author Timor Baasov Timor Baasov Department of Chemistry, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Yuval Shoham Corresponding Author Yuval Shoham Department of Food Engineering and Biotechnology, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Dietmar Schomburg Corresponding Author Dietmar Schomburg Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Klaus Hövel Klaus Hövel Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Dalia Shallom Dalia Shallom Department of Food Engineering and Biotechnology, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Karsten Niefind Karsten Niefind Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Valery Belakhov Valery Belakhov Department of Chemistry, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Gil Shoham Gil Shoham Department of Inorganic Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel Search for more papers by this author Timor Baasov Timor Baasov Department of Chemistry, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Yuval Shoham Corresponding Author Yuval Shoham Department of Food Engineering and Biotechnology, Technion, Haifa, 32000 Israel Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel Search for more papers by this author Dietmar Schomburg Corresponding Author Dietmar Schomburg Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany Search for more papers by this author Author Information Klaus Hövel1, Dalia Shallom2,4, Karsten Niefind1, Valery Belakhov3,4, Gil Shoham5, Timor Baasov3,4, Yuval Shoham 2,4 and Dietmar Schomburg 1 1Institute for Biochemistry, University of Cologne, Cologne, 50674 Germany 2Department of Food Engineering and Biotechnology, Technion, Haifa, 32000 Israel 3Department of Chemistry, Technion, Haifa, 32000 Israel 4Institute of Catalysis Science and Technology, Technion, Haifa, 32000 Israel 5Department of Inorganic Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2003)22:4922-4932https://doi.org/10.1093/emboj/cdg494 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info High-resolution crystal structures of α-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (β/α)8-barrel and a 12-stranded β sandwich with jelly-roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl—enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 Å apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion-like transition state via charge delocalization and specific protein—substrate interactions. The enzyme is an anti-protonator, and a 1.7 Å electrophilic migration of the anomeric carbon takes place during the hydrolysis. Introduction Most of the biomass synthesized by photosynthetic CO2 fixation is stored in the plant cell wall as polymeric carbohydrates, mainly in the form of cellulose and hemicellulose. The degradation of these polymers is a key step in the carbon cycle, and is mediated by microorganisms that produce specific enzymes, i.e. cellulases and hemicellulases (Figure 1A) (Shallom and Shoham, 2003). α-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that hydrolyze the arabinofuranosyl substitutions in hemicellulose. Some of these enzymes exhibit broad substrate specificity, acting on arabinofuranoside moieties at O-2 or O-3 as a single substituent, as well as from O-2 and O-3 doubly substituted xylans, xylooligomers and α-1,5-linked arabinans (Saha, 2000). The glycosidic bond between two sugars is one of the most stable bonds in nature, and its enzymatic hydrolysis, carried out by glycoside hydrolases, provides an acceleration rate (kcat/kuncat) which can be as high as 1017-fold (Wolfenden et al., 1998). The key elements in this remarkable catalysis are the finely tuned positions of the catalytic residues, and the distortion of the sugar ring so as to allow the stabilization of an oxocarbenium-ion-like transition state, as well as the movement of the hydrogen on the anomeric carbon to allow a direct nucleophilic attack (Zechel and Withers, 1999). Many glycosidases are modular proteins, and in addition to their catalytic domains include other functional modules, mainly carbohydrate-binding modules (CBMs) (Bourne and Henrissat, 2001). Currently, >8500 glycosidase sequences are known, and the sequence-based classification of their catalytic domains into glycoside hydrolase (GH) families and clans is available on the continuously updated Carbohydrate-Active Enzymes (CAZy) server (http://afmb.cnrs-mrs.fr/CAZY). The different bacterial, fungal and plant α-L-arabinofuranosidases are members of GH families 3, 43, 51, 54 and 62. Figure 1.(A) Upper, the basic structural components of xylan, and the hemicellulases responsible for its degradation; lower, the natural and synthetic substrates of α-L-arabinofuranosidases used in this work: Ara-α(1,3)-Xyl and 4-nitrophenyl-Ara. (B) The double-displacement reaction mechanism for retaining glycosidases (the Koshland mechanism). Download figure Download PowerPoint The enzymatic hydrolysis of glycosidic bonds results in either an overall inversion or retention of the anomeric configuration, and about two-thirds of the characterized GH families are retaining enzymes. Most retaining glycosidases cleave the glycosidic bond using two carboxylic acids, acting as a nucleophile and an acid/base. The hydrolysis proceeds through a two-step double-displacement mechanism, in which a covalent glycosyl—enzyme intermediate is formed amid the two reaction steps (Davies et al., 1998a; Sinnott, 1990) (Figure 1B). Crystal structures of native or mutant enzymes in complex with substrates, products, non-hydrolyzable substrate-analogues and transition-state analogues provide valuable detailed information regarding the specificity, binding mechanism and transition-state stabilization in retaining glycosidases, as recently reviewed by Vasella et al. (2002). The α-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA) belongs to the retaining GH-51 family, and its catalytic residues were recently identified, Glu175 is the acid/base, and Glu294 is the nucleophile (Shallom et al., 2002a,b). In this study, we describe high-resolution (1.2–2.0 Å) crystal structures of native and catalytic mutant of AbfA in complex with different substrates (Figure 1A). These structures include the Michaelis complexes with natural and synthetic substrates and the transient covalent arabinofuranosyl-enzyme intermediate with a non-fluorinated substrate. The structures allow thorough examination of the catalytic mechanism, including the two stable states of the glycosylation step, the interactions mediating substrate distortion and the features governing substrate specificity. Overall fold and oligomeric structure The crystal structure of the native AbfA was determined at 1.75 Å resolution (Table I). The structure contains all of the 502 amino acid residues, except the first four residues at the N-terminus, for which the electron density was not sufficiently clear. Figure 2 shows a representative electron density map demonstrating the quality and reliability of the model. The final refined models exhibit excellent stereochemistry and the bound ligand atoms refine well with average thermal factors comparable to the values for all protein atoms (Table I). Figure 2.Stereo view of the 2DFo − mFc electron density maps of a representative portion of the native AbfA at 1.75 Å, contoured at 2.0 σ. The area shown is that of the active-site residues Glu29 and Glu294, and the non-proline cis-peptide bond between residues Ala350 and Gln351. Selected hydrogen bonds between the protein and the water molecule are shown in dotted lines. Color coding: red, oxygen; blue, nitrogen; yellow, carbon. Download figure Download PowerPoint Table 1. Statistics of data collection, structure solution and refinement. Data set MAD Se peak MAD inflection MAD remote MIR Hg MIR Pt Native wild type 4-nitrophenyl-Ara E175A Covalent Ara E175A Ara-Xyl E175A Wavelength (Å) 0.97948 0.97971 0.9645 1.54178 1.54178 1.54178 0.84590 1.54178 1.54178 Space group R3 R3 R3 R3 R3 R3 R3 R3 R3 a,b (Å) 177 838 177 897 177 921 178 319 177 865 178 322 179 311 179 430 178 871 c (Å) 100 583 100 639 100 649 100 242 100 642 100 410 100 397 100 231 100 404 Unique reflections 135 163 131 318 132 006 65 700 72 203 119 711 370 260 75 814 107 887 Resolution range (Å) 20.0–1.68 20.0–1.68 20.0–1.68 20.0–2.10 20.0–2.05 20.0–1.75 50.0–1.20 20.0–2.0 20.0–1.80 Completeness (final shell) (%) 100.0 (99.9) 97.1 (98.4) 97.6 (98.7) 94.9 (96.0) 97.3 (96.9) 99.7 (99.7) 99.1 (97.9) 93.3 (96.2) 96.9 (92.4) / (final shell) 20.4 (5.9) 18.5 (3.4) 18.0 (3.5) 5.9 (3.1) 15.0 (2.9) 26.5 (2.8) 16.3 (1.7) 13.4 (1.7) 16.9 (1.9) Rsym (%) (final shell) 5.5 (27.0) 4.5 (35.7) 4.4 (31.1) 13.5 (29.5) 6.5 (31.8) 4.7 (39.5) 4.9 (38.3) 6.2 (37.5) 5.5 (41.0) FOM solve/resolve 0.57/0.81a 0.57/0.81a 0.57/0.81a 0.41/0.66b 0.41/0.66b R-factor (Rfree) 17.1 (20.4) 16.0 (17.9) 16.9 (21.5) 17.4 (21.3) Resolution range 20.0–1.75 50.0–1.20 20.0–2.00 20.0–1.80 R.m.s. deviations from ideality Bonds (Å) 0.014 0.014 0.018 0.013 Angles (°) 1490 1563 1667 1474 Average B-factors (Å)2 Main chain 23.5 15.9 40.9 28.9 Side chain 26.3 18.7 43.4 31.3 Solvent 37.8 33.3 48.2 39.2 Ligand 29.3 22.3 43.7 43.2 a MAD data to 2.5 Å resolution, before and after density modification. b Both derivatives to 3.0 Å resolution, before and after density modification. AbfA is a homohexamer with a D3 point symmetry (Hövel et al., 2003). The asymmetric unit of the R3 crystals contains two AbfA monomers with dimeric structure. Each AbfA monomeric subunit is organized into two domains: a (β/α)8 barrel (TIM barrel), and a 12-stranded β sandwich with a jelly-roll topology (Figure 3). The (β/α)8-barrel domain (residues 20–383) has an elliptical cross-section with the major axis of the distorted barrel running from β-strands 1 to 5, as observed for other (β/α)8 glycosidases (Jenkins et al., 1995). There are several deviations from the basic (β/α)8-barrel motif. An additional subdomain, composed of three α-helices and one anti-parallel β-sheet, is inserted between β-strand 2 and α-helix 2. Another α-helix is present between α-helix 3 and β-strand 4 and a short α-helix is inserted between β-strand 7 and α-helix 7. Furthermore, an anti-parallel β-sheet is found between β-strand 8 and α-helix 8. Figure 3.Overall structure of AbfA. (A) Two views of the AbfA monomer related by a 90° rotation. (B) The hexameric enzyme is shown along the crystallographic 3-fold axis. All non-schematic figures were prepared with Molscript (Kraulis, 1991), Bobscript (Esnouf, 1997) and Raster3D (Merritt and Bacon, 1997). Download figure Download PowerPoint GH families with overall similar fold can be further grouped into clans. Based on sequence analysis, the GH-51 arabinofuranosidases were classified as part of the GH-A clan, together with another 16 GH families (Henrissat et al., 1995; Zverlov et al., 1998). Within clan GH-A, the structures of enzymes from families 1, 2, 5, 10, 17, 26, 42 and 53 have been described, and their catalytic domains all share the same overall (β/α)8-topology and similar active site architecture. Thus, the (β/α)8 fold of AbfA, as well as the location of the catalytic residues (see below), are consistent with the classification of GH-51 within the GH-A clan. The second domain of AbfA is comprised of 12 β-strands with an uncommon jelly-roll topology. The eight β-strands of the jelly roll are arranged in two sheets that are packed against each other, stabilized by hydrophobic interactions between the upper and lower β-sheets. The eighth β-strand of the jelly roll is composed of the N-terminal residues 6–9, whereas the C-terminal residues 384–501 build up the remaining part of the domain. This domain shows structural similarity to domain C of several α-amylases and also to cellulose binding domains (Tormo et al., 1996; Fujimoto et al., 1998; Kamitori et al., 1999). It is possible that this domain formerly functioned as a carbohydrate-binding domain that lost its function during evolution. In this context it is worth noting that in our hands, AbfA did not exhibit binding ability to xylan (D.Shallom and Y.Shoham, unpublished data). The similarity to α-amylases relates also to the interactions between the jelly-roll domain and the (β/α)8-barrel. Several hydrogen-bonds are found between residues around the sixth and seventh α-helices of the (β/α)8-barrel, and residues from the adjacent sheet of the jelly roll domain. Similar interface architecture is found between domain C and the (β/α)8-barrel of the α-amylase from Thermoactinomyces vulgaris R-47, where it was proposed that the jelly-roll domain stabilizes the TIM-barrel structure (Kamitori et al., 1999). The oligomeric structure of AbfA can be described as a trimer of dimers. The hexameric architecture along the 3-fold axis is illustrated in Figure 3B. This molecular axis coincides with the crystallographic 3-fold axis resulting in a special position of the hexamer in the crystallographic unit cell. In the interface of each dimer, residues at the N-terminus of α-helix 5 and residues at the C-terminal end of α-helix 6 participate in the main interactions between the two monomers. The structural motifs that deviate from the basic (β/α)8-barrel and residues from α-helices 2, 3 and 4 of the barrel are all involved in the main interactions forming the hexamer. To date, only very few glycosidases have been suggested to be hexameric (Iriyama et al., 2000), and the structure of AbfA is the first crystal structure described for a hexameric glycosidase. The Michaelis complexes and substrate binding The kinetic characteristics of the acid/base E175A AbfA mutant have made it possible to obtain enzyme complexes with several substrates. For substrates with relatively poor leaving groups, such as 4-nitrophenyl-α-L-arabinofuranoside(4-nitrophenyl-Ara) and the natural substrate derived from xylan, arabinofuranosyl-α-(1,3)-xylose [Ara-α(1,3)-Xyl], the E175A mutant exhibits at least 103-fold lower activity than the wild-type enzyme (Shallom et al., 2002b). After soaking the crystal of the AbfA E175A mutant with each of the substrates 4-nitrophenyl-Ara and Ara-α(1,3)-Xyl, structures of the corresponding Michaelis complexes with the intact substrates were obtained at 1.2 and 1.8 Å resolution, respectively. The superimposition of the native enzyme structure with those of the Michaelis complexes reveals no significant change in the overall protein structure upon substrate binding (r.m.s.d. = 0.13 Å for all main chain atoms). Figure 4A shows the hydrogen bonds network at the active site of the wild-type enzyme. Interestingly, a glycerol molecule (originating from the cryo solution) is located in the active site, where the hydroxyls of the glycerol take similar positions to the hydroxyls of the substrates, as described below. Figure 4B and C shows the active site of the E175A mutant Michaelis complexes with the intact substrates 4-nitrophenyl-Ara and Ara-α(1,3)-Xyl, respectively. In both structures, the arabinofuranose moiety at the −1 subsite is bound by a large number of hydrogen bonds: each of its hydroxyls can form two possible hydrogen bonds with the enzyme. In the Ara-α(1,3)-Xyl complex, the xylose at the +1 subsite can form only one hydrogen bond and therefore is far less tightly bound than the arabinofuranose at the −1 subsite. This may explain the capability of AbfA to bind and hydrolyze substrates with structurally very different leaving groups (Shallom et al., 2002b). An additional electron density was observed in the +2 subsite, resulting from a second xylose connected to the O4 of the first xylose in some of the molecules, originating from the heterogeneity of the substrate (see Material and methods). The density map, however, was not clear enough, and the second xylose was not built into the final model. Figure 4.Interactions between ligands and key residues in the active site. Hydrogen bonds are shown as dotted black lines, their length in Ångströms is indicated. Color coding: yellow, carbon atoms of protein residues; green, carbon atoms of ligands; red, oxygen; blue, nitrogen. (A) Native AbfA with a glycerol molecule in the active site. The β-strands of the (β/α)8-barrel are shown schematically in light grey. (B) The Michaelis complex of the E175A mutant with 4-nitrophenyl-Ara. (C) The Michaelis complex of the E175A mutant with Ara-α(1,3)-Xyl. The insets in (B) and (C) show the two energetically close conformations of the arabinofuranose ring at the −1 subsite. (D) The covalent arabinofuranosyl—enzyme complex, obtained by using the E175A mutant with 2,5-dinitrophenyl-Ara. The inset shows the strong hydrogen bond between the sugar 2-hydroxyl and the nucleophile (Glu294). Download figure Download PowerPoint The crystal structures of the native and Michaelis complexes reveal nine key residues responsible for catalysis and substrate binding interactions: Glu29, Arg69, Asn74, Asn174, Glu175, His244, Tyr246, Glu294 and Gln351 (Figure 4). The location and function of many of these residues is conserved in clan GH-A (Sakon et al., 1996). The catalytic acid/base Glu175 is involved in a hydrogen bond to the conserved His244 at the end of β-strand 6 (not shown). In other GH-A clan enzymes, this position is occupied by homologous residues that can be a His or Tyr (family 5), Asn (families 1, 2, and 17), Asp (family 26), Gln (family 10), and a Ser (family 53) (Ryttersgaard et al., 2002). In AbfA, Asn174 preceding the proton donor at the end of β-strand 4 is involved in a hydrogen bond to the C2 sugar hydroxyl group of the arabinofuranosyl moiety (3.4 Å) (Figure 4C). This residue is invariant in the GH-A clan members and was shown to have a critical structural and functional role in maintaining the conformation and protonation state of the active site residues. For example, in the GH-5 family, a substitution of the homologous Asn by Asp led to nearly complete loss of enzyme activity (Navas and Beguin, 1992). Additionally, this residue seems to have an important role in assisting the formation of the covalent glycosyl—enzyme intermediate (Ryttersgaard et al., 2002). Arg69 of AbfA is located at the bottom of the active site (end of β-strand 2) and is conserved in families 1, 2, 5, 17 and 53 (not shown). This residue seems to keep the catalytic nucleophile Glu294 deprotonated, as Oϵ2 of Glu294 is within hydrogen bond distance to Nη1 of Arg69 (3.1 Å). In addition, Oϵ1 of Glu294 can form a hydrogen bond also with Tyr246, and this conserved residue can form two more hydrogen bonds, to the endocyclic O4 and to the O5 of the arabinofuranosyl at the −1 subsite. The mechanistic implications of these hydrogen bonds will be discussed later. A single non-proline cis-peptide bond is present in AbfA at the end of β-strand 8, between Ala350 and Gln351 (Figure 2). This unusual conformation constrains the orientation of the Gln351 side chain, thus enabling it to form a hydrogen bond with the 5-OH group of the arabinofuranosyl moiety (Figure 4B–D). This unique cis-peptide bond occurs in at least six different GH families with a (β/α)8-topology (1, 2, 5, 17, 18 and 42) (Sakon et al., 1996; Juers et al., 1999). Unlike AbfA, in most GH-A clan members an aromatic residue, often a tryptophan, forms the cis-peptide bond with the next residue. Similarly to AbfA, in these enzymes the cis-peptide bond constrains the position of the tryptophan and enables it to form hydrogen bonds to the substrate. The occurrence of this rare type of bond in the same location and for similar purposes in glycosidases with structurally very different substrates, gives further support for the proposed common evolutionary relationship between the glycosidases families sharing the (β/α)8-topology (Juers et al., 1999). The furanose 3-OH is within a hydrogen bond distance (2.9 Å) from the backbone nitrogen of Asn74 (Figure 4B). This residue is located above the C-terminal end of β-strand 2, in a similar position to a His residue, which is conserved in families 1, 5, 10 and 26. The His residue, absent in the structure of AbfA, is located at the end of β-strand 3 in the other GH-A clan members (Ryttersgaard et al., 2002), forming a hydrogen bond to the C3 hydroxyl of the glycosyl moiety. An intriguing feature of the active site in AbfA is Glu29 at the end of β-strand 1, which forms a direct contact with the substrate at subsite −1. Strong hydrogen bonds are observed between the Oϵ1 of Glu29 and the furanose C3 hydroxyls of the 4-nitrophenyl-Ara and Ara-α(1,3)-Xyl (Figure 4B and C). Replacing this residue in the GH-51 α-L-arabinofuranosidase from Thermobacillus xylanilyticus led to a reduction in the catalytic activity of three to four orders of magnitude, and it was therefore suggested to be a third catalytic residue (Debeche et al., 2002). From the current structure, it is clear that Glu29 is too far from the scissile bond to be directly involved in catalysis. However, it plays a key role in substrate binding: in addition to the strong hydrogen bond to the substrate, Glu29 can form hydrogen bonds to Asn74 and Gln351, which also participate in substrate binding. The elimination of Glu29 probably alters the hydrogen bonding network at the non-reducing end of the substrate, thus resulting in a severe drop in activity. Substrate specificity In this work we present the structures of a GH-A glycosidase in complex with a furanosidic substrate. The comparison of these structures to those of other GH-A glycosidases in complex with pyranosidic substrates can provide insight into the binding mechanism governing the substrate specificity of this related group of enzymes. The conformations of the arabinofuranose rings at the two Michaelis complexes of AbfA seem to be almost identical: a 4E conformation (an envelope with C4 above the plane) in the 4-nitrophenyl-Ara complex (Figure 4B), and the energetically close 4T0 conformation (an asymmetrical twist with C4 above and the endocyclic O4 beneath the plane) in the Ara-α(1,3)-Xyl complex (Figure 4C). These conformations place the glycosidic bond in a more quasi-axial orientation than in the E3 conformation (an envelope with C3 below the plane) of free L-arabinofuranoses, allowing an in-line attack of the nucleophile Glu294 at the anomeric carbon, and placing the glycosidic oxygen closer to the acid/base Glu175. In pyranose-specific retaining glycosidases, the distortion of the substrate from its relaxed 4C1 conformation is one of the most important characteristics of substrate binding, and therefore of catalysis itself (Tews et al., 1996; Davies et al., 1998b; Notenboom et al., 1998a; Sidhu et al., 1999; Vocadlo et al., 2001; Ducros et al., 2002). In furanoses, however, the energy barriers between the various envelope and twist conformations are small relative to those involved in the conformational inversion of pyranoses (Collins and Ferrier, 1995). The catalytic domain of AbfA shows resemblance to that of the endoglucanase Cel5A from Bacillus agaradhaerens. Cel5A is a retaining GH-5 family enzyme (grouped also as a clan GH-A glycosidase), which hydrolyzes β-1,4-glycosidic bonds in cello-oligosaccharides (Varrot and Davies, 2003). The structural similarity of AbfA and Cel5A made it possible to superimpose the structures, and compare the binding of glucopyranosidic substrates versus arabinofuranosidic substrates at the active site of these two GH families. The superposition of AbfA including its furanosidic substrate 4-nitrophenyl-Ara, together with the structure of the endoglucanase Cel5A with its pyranosidic substrate 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-D-cellobioside is shown in Figure 5. The D-glucose ring fits well in the active site of AbfA, except for the C6 and 6-OH, which appear to be too close to Trp298 (1.3 Å from the 6-OH to the tryptophan). This residue is conserved in most of the GH-51 arabinofuranosidases, but not in the only two GH-51 endoglucanases that hydrolyze glucopyranosydic substrates. Therefore, it appears that Trp298 is responsible for the discrimination against glucopyranosidic substrates in the GH-51 family. In a similar manner, the C5 and 5-OH of the L-arabinofuranose do not allow the binding in the endoglucanases active site, because of their close proximity to the conserved aromatic residue at the end of β-strand 8, Trp262 in Cel5A (1.8 Å from the 5-OH to the tryptophan). In AbfA, Gln351 is present in the equivalent position but, as described before, the preceding cis-peptide bond allows this residue to form a hydrogen bond to the 5-OH of the L-arabinofuranose. Thus, it appears that only two residues govern the distinction between D-glucopyranosidic and L-arabinofuranosidic substrates in these enzymes. It is tempting to speculate that the reciprocal replacements of these residues in the two enzymes will change their substrate specificity; experiments testing this hypothesis are currently under way. Figure 5.Substrate specificity of AbfA and Cel5A. Superposition of the Michaelis complexes of AbfA with 4-nitrophenyl-Ara and Cel5A endoglucanase with 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-D-cellobioside (PDB code: 1H2J). Only the catalytic residues and the residues responsible for the discrimination between the D-glucopyranosidic and L-arabinofuranosidic substrates are shown. Color coding: red, oxygen; blue, nitrogen; white, fluorine; yellow, carbon atoms of AbfA; light blue, carbon atoms of Cel5A. Download figure Download PowerPoint Interestingly, although belonging to the arabinofuranose-specific GH-51, AbfA can accommodate xylopyranosidic substrates. D-Xylopyranose and L-arabino furanose share spatial similarity, and the xylopyranoses do not have the C6 and 6-OH that cause the steric hindrance with Trp298 (as in glucose). This similarity rationalizes the existence of bifunctional α-L-arabinofuranosidases/β-D-xylosidases in GH-3 and GH-43 (Utt et al., 1991; Sakka et al., 1993; Lee et al., 2003). AbfA hydrolyzes the pyranosidic synthetic substrates aryl β-D-xylopyranosides with 2–30-fold lower activity (kcat) and 60–280-fold lower specificity (kcat/Km) compared with the corresponding aryl α-L-arabinofuranosides (Table II) (Shallom et al., 2002a,b). As mentioned earlier, the furanosidic substrates bind with relatively low distortion. Apparently, AbfA can accommodate xylopyranose in the active site, but without the necessary distortion required for the efficient catalysis of six-membered rings. This explains in part the lower specificity of AbfA towards the xylopyranosidic substrates. Table 2. AbfA kinetic parameters for the hydrolysis of aryl-α-L-arabinofuranosides and aryl-β-D-xylopyranosidesa Phenol substituent (pKa) Glyconb kcat (s−1) Ratio kcat [AF/Xyl] Km (mM) Ratio Km [AF/Xyl] kcat/Km (s−1 mM−1) Ratio kcat/Km [AF/Xyl] 2,5-Dinitro (5.15) AF 190 2.6 0.35 1/23 540 60 Xyl 74 8.0 9.3 3,4-Dinitro (5.36) AF 340 34 0.53 1/8 640 280 Xyl 10 4.4 2.3 4-Nitro (7.18) AF 87 4.3 0.65 1/23 130 100 2-Nitro (7.22) Xyl 20 15.3 1.3 a Values taken from Shallom et al. (2002a,b). b AF, arabinofuranosyl; Xyl, xylopyranosyl. Trapping t
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