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Crystal Structure of Exo-Inulinase from Aspergillus Awamori: the Enzyme Fold and Structural Determinants of Substrate Recognition

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Crystal Structure of Exo-Inulinase from Aspergillus Awamori: the Enzyme Fold and Structural Determinants of Substrate Recognition doi:10.1016/j.jmb.2004.09.024 J. Mol. Biol. (2004) 344, 471–480 Crystal Structure of Exo-inulinase from Aspergillus awamori: The Enzyme Fold and Structural Determinants of Substrate Recognition R. A. P. Nagem1†, A. L. Rojas1†, A. M. Golubev2, O. S. Korneeva3 E. V. Eneyskaya2, A. A. Kulminskaya2, K. N. Neustroev2 and I. Polikarpov1* 1Instituto de Fı´sica de Sa˜o Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked Carlos, Universidade de Sa˜o b-D-fructofuranose residues in inulin, levan and sucrose releasing Paulo, Av. Trabalhador b-D-fructose. We present the X-ray structure at 1.55 A˚ resolution of Sa˜o-carlense 400, CEP exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase 13560-970, Sa˜o Carlos, SP family 32, solved by single isomorphous replacement with the anomalous Brazil scattering method using the heavy-atom sites derived from a quick cryo- soaking technique. The tertiary structure of this enzyme folds into 2Petersburg Nuclear Physics two domains: the N-terminal catalytic domain of an unusual five-bladed Institute, Gatchina, St b-propeller fold and the C-terminal domain folded into a b-sandwich-like Petersburg, 188300, Russia structure. Its structural architecture is very similar to that of another 3Voronezh State Technological member of glycoside hydrolase family 32, invertase (b-fructosidase) from Academy, pr. Revolutsii 19 Thermotoga maritima, determined recently by X-ray crystallography The Voronezh, 394017, Russia exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87 A˚ , reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition. q 2004 Elsevier Ltd. All rights reserved. Keywords: exo-inulinase; Aspergillus awamori; glycoside hydrolase; crystal- *Corresponding author lographic structure; X-ray structure Introduction Jerusalem artichoke (Helianthus tuberosus)and chicory (Cichorium endivia).3 Levan, a similar poly- Most plants store starch or sucrose as reserve fructan, which consists of linear b-(2,6)-linked carbohydrates; however, about 15% of flowering fructofuranosyl units, is produced in dicotyledo- plant species store fructans,1 which are branched nous species, and is found in several types of grass, polymers of fructose. Inulin is a widespread plant e.g. Dactylis glomerata.4 Mixed levan, consisting of polyfructan that has linear chains of b-(2,1)-linked both 2/1 and 2/6 linked b-D-fructosyl residues, is fructose residues attached to a terminal sucrose found in most plant species belonging to the Poales residue.2 Inulin serves as a storage polysaccharide family, including wheat and barley.4,5 in the Compositae and Gramineae, and is accumu- Inulin, due to its role as a relatively inexpensive and lated in the underground parts of several plants abundant substrate for production of rich fructose of the Asteracea, including Vernonia herbacea, syrups, is of considerable industrial interest.3,6 This type of syrup may be obtained by acid hydrolysis 8 † R.A.P.N. and A.L.R. contributed equally to this work. of inulin at 80–100 C; however, under these conditions fructose easily degrades, producing Abbreviations used: EI, exo-inulinase; GHF, glycoside 7 hydrolase family; Bsl, Bacillus subtilis levansucrase. colored products, such as difructose anhydrides. E-mail address of the corresponding author: The enzymatic hydrolysis with inulinases and [email protected] exo-inulinases (EI) offers an alternative approach to 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. 472 A. awamori Exo-inulinase 3D Structure produce fructose free of undesirable by-products. form and was refined to the final Rwork and Rfree of Successful biotechnological applications of inulinases 17.0% and 19.8%, respectively. The model has good have brought significant attention to these enzymes.8 stereochemical parameters, with root-mean-square Inulinases expressed by Penicillium,9–11 Kluyvero- deviations (rmsd) of 0.007 A˚ for bond distances, myces,12 and Aspergillus13,14 are the most intensively and 1.178 for bond angles. The crystallographic studied fungal enzymes of this class. Exo-inulinases model was also refined in the orthorhombic crystal ˚ (EC 3.2.1.80, also known as fructan b-fructosidase or form at 1.89 A resolution, with a final Rwork and exo-b-D-fructosidase) hydrolyze terminal, non- Rfree of 16.5% and 19.9%, respectively (rmsd of reducing 2,1-linked and 2,6-linked b-D- 0.011 A˚ for bond distances and 1.318 for bond fructofuranose residues in fructans, with concomi- angles). These structures were found to be essen- tant release of b-D-fructose. The natural substrates tially the same, with the exception of the degree of of exo-inulinases include inulin, levan and glycosylation. The crystallographic structure of sucrose. Nucleotide and amino acid sequences EI:fructose complex was refined at 1.87 A˚ resolution 15 were reported for exo-inulinases from yeast, with final Rwork and Rfree of 14.5% and 16.9%, 16 14 chicory, Aspergillus awamori and Geobacillus respectively. All residues but one fall in the allowed 17 stearothermophilus. regions of the Ramachandran plot, as determined Based on amino acid sequence comparisons, it by PROCHECK.22 Ser348 lies outside of the allowed has been demonstrated that the exo-inulinase (EI) regions, probably because its Og forms hydrogen from A. awamori belongs to the glycoside hydrolase bonds with both Asn333 Nd2 (2.80 A˚ ) and Trp331 18 family (GHF) 32. This family includes invertase, N31 (3.15 A˚ ), which results in energetically unfavor- inulinase, levanase and two types of 1-fructosyl able conformation of this residue. transferase. The catalytic activity of the enzymes The enzyme folds into two domains (Figure 1). from GHF 32 is based on a similar mechanism that The N-terminal domain, containing the catalytic involves overall retention of the configuration at site, comprises 353 amino acid residues (Phe20 to the anomeric carbon atom of the substrate. The Gln372). It belongs to the b-propeller fold with five similarities of the amino acid sequences and activity b-sheets arranged like blades of a propeller, radially toward the fructose containing poly- and oligo- and pseudosymmetrically around a central axis. saccharides possibly reflect an evolutionary Each b-sheet contains four antiparallel b-strands. relationship among GHF 32 members. Strands 1, 2, 3, and 4 are connected by hairpin turns, Here, we describe the first crystal structure of and strand 4 of one sheet is connected to strand 1 of exo-inulinase in a ligand-free and fructose-bound the next. Strand 1, in each “blade” of the b-propeller, form, and discuss a structural basis of the catalytic is the closest to the central axis and runs parallel mechanism of the enzyme. with it. The overall shape of the domain can be described as cylindrical, with strand 4 of each blade being on the outside of the cylinder. Results and Discussion The five-bladed b-propeller fold is rare, and is exemplified by tachylectin-2,23 Cellvibrio japonicus 24 Quality of the model and a three-dimensional a-L-arabinanase A43 and Bacillus subtilis levansu- structure of exo-inulinase crase.25 Remarkably, the latter two enzymes are members of GHF 43 and 68, respectively. Common Two crystal forms of the native exo-inulinase sequence motifs have been identified between GHF were obtained under the same crystallization 32 and 68,26 as well as between GHF 68 and 4327 conditions. Crystallization of the enzyme in the despite low (w15%) sequence identity between primitive orthorhombic P212121 space group has these families. been reported.14 The new crystal form belongs to The EI C-terminal domain, comprising 156 amino the monoclinic P21 space group. The crystallo- acid residues (Arg382 to Asn537), consists of graphic structure of the enzyme was solved by the two b-sheets, each composed of six b-strands, SIRAS method, using a quick cryo-soaking tech- assembled into a sandwich-like structure. The first nique,19–21 at 1.55 A˚ resolution in the monoclinic and second domains are connected by a short Figure 1. (a) Ribbon represen- tation of the secondary structure elements of exo-inulinase. The N- terminal domain belongs to the five-bladed b-propeller fold. Each blade is shown in a different color (blade 1 is yellow, blade 2 is marine, blade 3 is red, blade 4 is lime and blade 5 is orange). The second domain, colored in slate, consists of 12 b-strands arranged in a b-sandwich fold. The short polypeptide chain connecting the two domains is depicted in pink. The five N-linked oligosaccharides present in the orthorhombic crystal are represented as stick. (b) Representation of exo-inulinase surface with the fructose molecule in the activity site. A. awamori Exo-inulinase 3D Structure 473 polypeptide linker, with relative positions and although the amino acid sequences of these orientations of the domains stabilized by multiple enzymes hardly bear any similarity (amino acid hydrogen bonds and hydrophobic interactions. sequence identity is only 12%), the fold of both Comparison of the present EI model with the protein domains is very similar with rmsd between structures deposited with the Protein Data Bank, 105 Ca atom pairs of 1.97 A˚ (Figure 2(b)). Overall, carried out using the DALI server,28 reveals a the fold of EI is essentially the same as that of significant structural similarity between the EI Thermotoga maritima invertase, another member of N-terminal b-propeller domain and the N-terminal GHF 32, which has been determined very recently domain of levansucrase from B.
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