Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes Ingunn A Hoell1, Bjørn Dalhus2, Ellinor B Heggset1, Stein I Aspmo1 and Vincent G H Eijsink1 ˚ Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, As, Norway Institute of Medical Microbiology, Section for Molecular Biology, National University Hospital, Oslo, Norway Keywords ChiG; chitinase; family 19; Streptomyces coelicolor A3(2); subsite structure Correspondence V G H Eijsink, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, ˚ PO Box 5003, 1432 As, Norway Fax: +47 64965901 Tel: +47 64965892 E-mail: vincent.eijsink@umb.no (Received June 2006, revised September 2006, accepted September 2006) doi:10.1111/j.1742-4658.2006.05487.x We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2) Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin–remazol–brilliant violet) and a small, presumably amorphous, subfraction of a-chitin and b-chitin, but was not capable of degrading crystalline chitin completely The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol 358, 472–484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites () to + 2), as opposed to six () to + 3) for plant enzymes The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates The importance of these two residues for catalysis was confirmed by a sitedirected mutagenesis study Chitinases (EC 3.2.1.14) are glycoside hydrolases that catalyze the hydrolysis of chitin, a carbohydrate polymer of 1,4-b-linked GlcNAc Chitin is found in the cuticle of insect shells, in shells of crustaceans, and in the cell walls of many fungi, making chitin the second most abundant polysaccharide in nature after cellulose [1,2] Chitinases are present in a wide variety of organisms, such as bacteria, viruses, higher plants and animals [1–4] The hydrolysis products of chitin, chitooligosaccharides, are of interest in several biological and biotechnological processes [1,2] Glycoside hydrolases are divided into different families based on primary sequence, three-dimensional structure, and catalytic mechanism [5,6] Family 18 and family 19 glycoside hydrolases both contain chitinases Members of the two families have very different three-dimensional structures and use different catalytic mechanisms The catalytic domains of family 18 chitinases have a (b ⁄ a)8 fold [6] and use a substrate-assisted double-displacement mechanism, which leads to retention of the configuration of the anomeric carbon [7,8] The catalytic domains of family 19 chitinases have Abbreviations ChiC, chitinase C from Streptomyces griseus HUT6037; ChiF, chitinase F from Streptomyces coelicolor A3(2); ChiG, chitinase G from Streptomyces coelicolor A3(2); CM-chitin RBV, carboxymethyl-chitin–remazol–brilliant violet; 4-MU, 4-methylumbelliferyl; TEV-protease, tobacco etch virus NIa protease FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4889 Bacterial family 19 chitinase I A Hoell et al high a-helical contents and share some structural similarity with chitosanases and lysozyme [9,10] Family 19 chitinases use a single-displacement mechanism, which leads to inversion of the configuration of the anomeric carbon [6,11] The catalytic mechanism of family 19 chitinases has been studied in detail by modeling [12], but experimental studies that underpin the proposed mechanism are remarkably scarce [13] In fact, in the CAZY database of glycoside hydrolases [5] (http:// afmb.cnrs-mrs.fr/CAZY/), the catalytic proton donor and the catalytic base are not annotated Family 19 chitinases are commonly found in many plants, but were only recently discovered in bacteria The first bacterial family 19 chitinase, chitinase C (ChiC), was found in Streptomyces griseus HUT6037 in 1996 [14] Subsequently, several bacterial family 19 chitinases have been identified, including chitinases from Burkholderia gladioli, Vibrio cholerae, Haemophilus influenzae, and Pseudomonas aeruginosa Although many plant family 19 chitinases are known, crystal structures are available for only two of these [9,15–17] The first structure of a bacterial family 19 chitinase has just recently been solved (Streptomyces griseus HUT6037 ChiC; PDB accession code 1WVU) [18] Streptomyces coelicolor A3(2) is a spore-forming soil-borne Gram-positive bacterium that grows via a branching mycelium, mainly by tip growth [19] The genome was fully sequenced in 2002, and this revealed that the ability of S coelicolor A3(2) to exploit nutrients in the soil is associated with the ability to produce many different hydrolases, including 13 chitinases [20] Among these chitinases we find two putative family 19 enzymes, ChiF and ChiG [21,22], which share 84% identity to each other, and 80% and 75% identity to the catalytic domain of ChiC from Streptomyces griseus, respectively [21] ChiF has a similar domain structure to ChiC, consisting of a catalytic domain and an N-terminal chitin-binding domain ChiG, on the other hand, lacks this chitin-binding domain, and consists only of a catalytic domain [22] The chiG gene encodes a 244 amino acid chitinase, including a 29 amino acid leader peptide sequence The closest relative of ChiG among the two plant family 19 chitinases with known crystal structures comes from Canavalia ensiformis (Jack beans; 37% sequence identity) Interestingly, ChiG and most other bacterial chitinases seem to have different catalytic centers from the plant enzymes, as there is a 13-residue deletion between the putative catalytic residues, thus making them closer in sequence in the former (Glu68 and Glu77 in ChiG; Fig 1) Inspection of the structures of the two plant enzymes shows that this deletion is located on a loop near the (putative) + subsite [9,12] Sequence align4890 ments (Fig 1) show several other deletions in the bacterial enzymes that potentially could affect interactions with the substrate In order to provide more insight into family 19 chitinases in general and into the differences between plant and bacterial enzymes in particular, we have overexpressed ChiG from S coelicolor A3 (2) in Escherichia coli and characterized the enzyme with respect to its catalytic properties and crystal structure The crystal structures of ChiG and ChiC [18] permitted structural comparison between bacterial and plant enzymes, which provided a structural explanation for observed differences in enzymatic properties The role of the putative catalytic residues was confirmed by site-directed mutagenesis Results Enzymology Overexpression of ChiG in E coli BL21Star (DE3) yielded soluble and active enzyme, which could easily be purified by Ni-affinity chromatography (supplementary Fig S1; typical yields were in the range 2–7 mg of ChiG per liter of culture) Tests with several substrates showed that removal of the His tag by tobacco etch virus NIa protease (TEV-protease) did not affect the catalytic properties of the enzyme ChiG did not show any activity against 4-methylumbelliferyl (4-MU)-(GlcNAc)2 or 4-MU-(GlcNAc)3, as also earlier demonstrated by Saito et al [21] On the other hand, ChiG showed activity against a-chitin and b-chitin (supplementary Fig S2), chitooligosaccharides (Figs and 3), carboxymethyl-chitin–remazol–brilliant violet (CM-chitin RBV) and also against chitosan (E Hegg˚ set, I A Hoell & K M Varum, unpublished results) Figure shows the kinetics of product formation during oligosaccharide degradation Specific activities (derived from initial substrate disappearance rates, Fig 2) were 1.13 ± 0.07 lmolỈs)1Ỉmg)1, 0.63 ± 0.03 lmolỈs)1Ỉmg)1, 0.49 ± 0.05 lmolỈs)1Ỉmg)1, and 0.08 ± 0.01 lmolỈs)1Ỉmg)1 for (GlcNAc)6, (GlcNAc)5, (GlcNAc)4 and (GlcNAc)3, respectively Degradation of both a-chitin and b-chitin yielded (GlcNAc)2 and (GlcNAc)3, whereas after long incubation times (24 h) significant amounts of GlcNAc were observed, due to further degradation of (GlcNAc)3 to (GlcNAc)2 and GlcNAc (results not shown) The specific initial activities towards a-chitin and b-chitin (judged from a short initial linear phase in product formation) were 0.09 ± 0.01 lmolỈs)1Ỉmg)1 and 0.11 ± 0.03 lmolỈs)1Ỉmg)1, respectively However, only a minor fraction of the chitin was degraded at these speeds; the reactions rapidly slowed down and a larger part of FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS I A Hoell et al Bacterial family 19 chitinase Fig Structure-based multiple sequence alignment of all family 19 chitinases with known structure The figure shows two plant enzymes, from Hordeum vulgare (barley [9]) and Canavalia ensiformis (Jack bean [17]), and two bacterial enzymes, chitinase C (ChiC) (PDB accession code 1WVU) from Streptomyces griseus HUT 6037 (catalytic domain only [18]) and chitinase G (ChiG) The alignment was made using the protein structure comparison service SSM at the European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/ssm [45]) Four major deletions in the bacterial enzymes are indicated by A, B, C and C-term, and the 161–166 loop (numbering of the barley enzyme; see text) is indicated by dots above the sequence Residues involved in disulfide bridge formation are marked with closed or open bullets, for conserved and nonconserved bridges, respectively The closed triangles indicate two conserved glutamate residues involved in catalysis Fully conserved secondary structure assignments are indicated with h for a-helix and s for b-strand The consensus helix comprising residues 169– 177 in ChiG is extended towards Cys183 in the other three enzymes the substrates remained undegraded, even after prolonged incubation Thus, ChiG is much less effective than e.g the two-domain family 18 chitinase ChiA from S marcescens which can degrade b-chitin completely (supplementary material Fig S2) Yields were lowest for a-chitin, whereas ChiA is capable of almost completely degrading this substrate too, albeit at a slower rate [23] Figure shows that the tetramer is exclusively converted to two dimers, meaning that there is only one binding mode for this substrate The longer oligomeric substrates have several potentially productive binding modes Preferred binding modes can be analyzed by determining anomeric ratios of products formed early during the reaction [24–26] The results (Fig 3, Table 1) show that during degradation substrates stayed close to the expected equilibrium ratio of 60% a-anomer and 40% b-anomer, whereas all products had anomer ratios that were close to 80 : 20 One would expect an 80 : 20 ratio: (a) if the enzyme is inverting (that is, each new reducing end has an a-anomeric configuration, as would be expected for a family 19 enzyme [11]); and (2) if each product contains a 50 : 50 mixture of newly formed (100% a) and existing (60% a) reducing ends Figure 2A shows that (GlcNAc)6 was hydrolyzed to (GlcNAc)4 + (GlcNAc)2 and (GlcNAc)3 + (GlcNAc)3 The efficiency of the first reaction was about double that of the second reaction (see legend to Fig 2) The 80 : 20 anomeric distribution in the tetramer and dimer fractions (Fig 3, Table 1) shows that the first reaction equally often results from cleavage between sugars and (new reducing end on the tetramer product) as from cleavage between sugars and (new reducing end on the dimer product) Thus, the hexamer is degraded through three types of productive binding modes with approximately similar frequencies, leading to cleavage after sugars 2, or Hydrolysis of (GlcNAc)5 initially FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4891 Bacterial family 19 chitinase I A Hoell et al Fig Time course of the degradation of chitooligosaccharides by chitinase G (ChiG) (A) Hexamer (B) Pentamer (C) Tetramer (D) Trimer (hexamer), } (pentamer), h (tetramer), n (trimer), X (dimer) and s The concentrations of the various oligosaccharides are indicated by (monomer) All reactions were run under identical conditions, except for the reaction with trimer, in which the enzyme concentration was increased 50-fold In (A), note that a single cleavage can produce two trimers or one dimer + one tetramer; the graph thus shows that the reaction producing tetramer + dimer happens about twice as often as the trimer-producing reaction In the reactions depicted in (A) and (B), monomers were only detected after prolonged incubation, i.e after depletion of the original substrate With the tetramer (C), monomers were never detected yielded equal amounts of (GlcNAc)3 and (GlcNAc)2; the 80 : 20 anomeric ratio of the products indicates that cleavage after sugar or sugar occurs approximately equally often Structure The overall structure of ChiG (Fig 4, supplementary Fig S3) is similar to that of the family 19 chitinase from barley, the best studied of the plant chitinases [9,12,13,27,28] and essentially identical to that of the catalytic domain of ChiC from S griseus HUT6037 ˚ ([18]; rms 0.84 A) The only notable difference between the two bacterial enzymes occurs in the 178–183 region: the consensus helix comprising residues 169– 177 in ChiG (Fig 1) is extended towards Cys183 in ChiC (and in the plant enzymes) Compared to the barley enzyme, ChiC and ChiG lack three loops (A, B and C) and a C-terminal extension (Figs and 4B) In addition, one other loop, 4892 comprising residues 161–166 in the barley enzyme, has ˚ shifted its position by up to A (Fig 4B) Two of the three disulfide bridges found in the plant enzymes are conserved (in ChiG: Cys87–Cys95 and Cys183– Cys215), whereas the third bridge is lacking, due to the deletion of the A-loop (Fig 1) The enzyme has a deep groove that is likely to bind the substrate [6,12] and that contains the putative catalytic residues Glu68 and Glu77 (Figs and 4) For an inverting enzyme, one would expect the distance between the carboxyl ˚ oxygens of these two glutamates to be about 10 A [6] ˚ In ChiG, this distance is 9.5 A for the closest pair of oxygens The four major deletions in ChiG (loops A, B and C and the C-terminus) as well as the one major structural difference (161–166 loop) compared to the plant enzymes can be divided into two subsets of interrelated changes, with each subset affecting one side of the substrate-binding groove of the enzyme (Fig 4B,C) On the side where the nonreducing end of the substrate FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS I A Hoell et al Bacterial family 19 chitinase Fig HPLC analysis of reaction mixtures under conditions preventing anomeric equilibrium The top panel represents a standard mixture of GlcNAc oligomers showing the standard 60 : 40 ratio between the a-anomer and the b-anomer at equilibrium The other panels show the results of partial hydrolysis of (GlcNAc)6 and (GlcNAc)5 by chitinase G (ChiG) In these panels, substrates display the 60 : 40 ratio, whereas the ratios for the products are close to 80 : 20 See text and Table for details The small peaks close to the tetramer position in the chromatogram for (GlcNAc)5 and close to the pentamer position in the chromatogram for (GlcNAc)6 were also present in control samples and are not due to enzyme action Table Anomeric configuration in the reaction mixtures depicted in Fig Hydrolysis of (GlcNAc)6 Products (GlcNAc)2 (GlcNAc)3 (GlcNAc)4 Substrate Hydrolysis of (GlcNAc)5 a-Anomer (%) a-Anomer (%) reduced ability to bind sugars in the ) and ) positions On the side where the reducing end of the substrate binds, the interacting loops A and B in the barley enzyme extend the substrate-binding surface beyond subsite + 2, primarily through the exposed Trp72 The importance of tryptophans in positions such as Trp72 for the efficiency of chitinolytic enzymes is well established [29] There is another Trp at position 82 in loop B, which is shielded from solvent in the barley enzyme (Fig 4C), but which is more exposed in the absence of Trp72, as in the jack bean enzyme ChiG lacks loops A and B, and thus seems to have reduced ability to bind sugars beyond subsite + In addition, Thr69, thought to be important for sugar binding in subsite + of the barley enzyme, is not conserved and is replaced by Gly in ChiG No structural data were obtained for the 11 N-terminal residues of ChiG Compared to the barley and the jack bean enzymes, ChiG contains an N-terminal extension of eight and seven residues respectively (Fig 1) The N-termini of the plant enzymes and the first residue in the ChiG structure (Phe12) (Fig 4A), are located on the opposite side of the enzyme to the catalytic center and the substrate-binding groove The same applies to the structurally observed N-terminus of the catalytic domain of ChiC, which corresponds to residue in ChiG [18] In ChiC, this N-terminus is part of a linker (with unknown structure) that connects an N-terminal chitin-binding domain to the catalytic domain All in all, it is highly unlikely that the N-terminal extensions in ChiG directly affect catalytic architecture 78 79 80 62 b-Anomer (%) 22 21 20 38 81 79 – 62 b-Anomer (%) 19 21 – 38 binds, loop C and the C-terminal extension affect the position of the 161–166 loop, which contains two polar side chains (Gln162 and Lys165) thought to be important for sugar binding in subsites –3 and –4 (in the barley enzyme [12,27]) In ChiG, in the absence of the C-loop and the C-terminal extension, the 161–166 loop has moved toward the catalytic center In addition, the Gln and Lys residues have been replaced by Thr (Thr153 and Thr157) Thus, ChiG seems to have a Mutagenesis of the catalytic center Figure shows that two glutamates thought to make up the catalytic center in the barley family 19 chitinase [9,13] are conserved in ChiG, despite the large deletion in between these two residues The role of these glutamates was demonstrated by site-directed mutagenesis Table shows that all mutants had greatly reduced catalytic activity Some detectable activity was still left upon mutation of Glu77 (2000–6000-fold reduction in activity), whereas mutation of Glu68 reduced activity to below the level that could be detected with our assays (> 24 000-fold reduction in activity) Discussion Whereas family 19 chitinases are widespread in higher plants, their occurrence in prokaryotes has only recently been discovered [14,22,26,30] Judged by avail- FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4893 Bacterial family 19 chitinase I A Hoell et al B A C Fig Structure of chitinase G (ChiG) and comparison with the barley chitinase (A) Cartoon showing the overall fold of ChiG with transparent surface The side chains of the catalytic residues, Glu68 and Glu77 are shown in red (B) Structural superposition of ChiG and the barley enzyme The picture shows a cartoon of the barley enzyme (PDB accession code 2bba; cyan) and the surface of ChiG, with the view being rotated 90° relative to (A) (the view is into the substrate-binding groove) Important structural elements are labeled (see text for details), and the catalytic glutamates are shown in red (C) Differences between the barley enzyme (cyan, left) and ChiG (blue, right) in the substrate-binding cleft The side chains of the catalytic residues are shown in green The side chains of residues that are deleted (Trp72, Trp82), mutated (Thr69 ⁄ Gly70) or mutated and relocated (Lys165 ⁄ Thr157 and Gln162 ⁄ Thr153) in ChiG are shown in red The side chains of four fully conserved residues in subsites ) (right side) to + (left side) are shown in purple Note that the ) and ) subsites partly consist of backbone atoms [12]; these are structurally well conserved, but not shown in the picture The pictures were made with PYMOL [47] able sequences, some bacterial family 19 chitinases have catalytic domains that are at least as large as those of the plant enzymes and that may contain at least six subsites [26,30] However, the catalytic 4894 domains of ChiG, ChiC and most other known bacterial family 19 chitinases are smaller than those of the plant enzymes Unfortunately, there is no direct structural information concerning the interaction between FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS I A Hoell et al Bacterial family 19 chitinase Table Specific activity of site-directed mutants of ChiG towards (GlcNAc)6 Enzyme Specific activity towards (GlcNAc)6 (lmolỈmg)1Ỉs)1) Relative specific activity Wild-type ChiG E68Q E68A E77Q E77A 1.13 ± 0.07 Not detectable Not detectable 0.00057 0.00019 < 4.2 · 10)5a < 4.2 · 10)5a 5.0 · 10)4 1.7 · 10)4 a Estimated on the basis of the approximate detection limit of the assay family 19 chitinases and their substrates Soaking experiments were not successful and nor were cocrystallization experiments with the inactive mutant E68Q [9] (B Dalhus, S I Aspmo & I A Hoell, unpublished results) However, the interaction between the barley chitinase and (GlcNAc)6 has been studied in great detail by computational techniques, exploiting the (limited) structural similarity between family 19 chitinases and lysozyme [9,12,27,31]; (the crystal structure of a lysozyme–(GlcNAc)3 complex was used for modeling purposes) By analogy to lysozyme, these studies assumed the presence of six subsites, running from ) to + [subsites are numbered according to standard nomenclature; cleavage occurs between the sugar units bound in subsites ) and + [32]; (note that in the older literature, these subsites are referred to as A () 4) to F (+ 2)] Judging by the structure of the barley enzyme, one would assume that there is affinity for the substrate beyond the + subsite, primarily because of the prominent Trp residue at position 72, ˚ approximately 15 A from the catalytic center This Trp would be able to interact with sugars bound at positions + and + Indeed, analysis of the hydrolysis of (GlcNAc)6 by barley chitinase [28] and by a highly similar chitinase from rice [25] led to the conclusion that these enzymes have a + subsite with considerable affinity for a sugar moiety All residues thought to be involved in sugar binding at the ) to + subsites in the barley enzyme are fully or, at least functionally, conserved in ChiG (Fig 4C), except for Thr69 in the + subsite, which is replaced by a glycine Beyond this central region, ChiG clearly differs from the barley enzyme, as a consequence of the loop deletions and the resulting conformational change in the 161–166 loop The deletion of the Trp72-containing loop (loop B in Fig 4B) removes putative subsites + and + 4, whereas the conformational change of and the mutations in the 161–166 loop remove putative subsites ) and ) [12,27] Thus, in ChiG, the substrate-binding groove ⁄ surface is less extended and does not seem to contain more than the four central subsites The presence of only four subsites was confirmed by studies on the degradation of pentamers and hexamers (Fig 2, Table 1) For example, productive binding of the hexamer by ChiG occurs in three different binding modes (in ‘subsites’ ) to +2, ) to +3 and ) to +4) with almost identical frequencies This shows that there is little binding affinity in subsites beyond ) and + The barley and rice enzymes show clearly different product profiles, primarily due to the presence of a + subsite [25,28] Most interestingly, whereas ChiG hydrolyzed tetrameric, pentameric and hexameric substrates with rather similar rates (varying less than 2.5-fold), the efficiency of the barley enzyme is strongly dependent on substrate length Studies by Hollis et al [27] showed that the barley enzyme degrades the hexamer about 200 times faster than the tetramer This confirms that the barley enzyme and ChiG have different catalytic properties, in accordance with the observed structural differences Using structural information only, Kezuka et al [18] have hypothesized that ChiC has six subsites, namely subsites ) to + 2, as in hen egg-white lysozyme This hypothesis is not confirmed by the present analysis of enzymatic properties of ChiG, or by our analysis of the ChiG and ChiC structures Despite the deletion of the B-loop, the two putative catalytic residues Glu68 and Glu77 are structurally well conserved between ChiG and the plant enzymes Andersen et al [13] have previously shown that the corresponding residues in the barley enzyme, Glu67 and Glu89, are essential for catalysis The mutagenesis studies presented here show that Glu68 and Glu77 are essential for catalysis by ChiG Mutation of Glu68 to Gln resulted in total inactivation, whereas mutation of Glu77 did not This is in accordance with the notion that Glu68 is the catalytic acid, whereas Glu77 is the catalytic base [6,13] The activity of ChiG towards chitooligosaccharides was found to be comparable to that of other chitinases, including, for example, well-known family 18 exochitinases and endochitinases from Serratia marcescens [23] ChiG showed relatively high initial activity towards chitin, but the overall ability to degrade the polymer was limited, as compared to, for example multidomain family 18 chitinases from S marcescens [33] (supplementary Fig S2) Thus, while ChiG is rather active towards soluble substrates and some (amorphous) regions of insoluble chitin, the enzyme is not very efficient in degrading crystalline polymeric substrates Other bacterial family 19 chitinases such as FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4895 Bacterial family 19 chitinase I A Hoell et al ChiC and ChiF contain an additional substrate-binding domain, which could make these enzymes more efficient with crystalline substrates However, deletion of this domain had only a modest effect on enzyme efficiency with crystalline chitin [34] Like ChiG, both ChiC and ChiF display relatively high activities towards noncrystalline chitin forms, and low activities towards crystalline chitin [22,35] It has previously been shown that chitinases with high activity towards crystalline chitin, such as Bacillus circulans chitinase A1 and S marcescens chitinase A, have extended substrate-binding grooves (at least six subsites) Notably, these grooves contain a stretch of linearly aligned aromatic residues that play an important role in guiding a chitin chain from the crystalline chitin surface to the catalytic center [29] Our finding that the bacterial enzymes have only four subsites and the absence of aromatic residues in these subsites may explain why ChiG and related enzymes have low activity towards crystalline chitin The open active site of ChiG suggests that ChiG binds polymers in an endo-fashion This was confirmed by the observation that hydrolysis of chitin led to significant production of trimers during degradation of both a-chitin and b-chitin (exoenzymes tend to almost exclusively produce dimers) Studies on the degradation of colloidal chitin by ChiC led to a similar conclusion [14] Most probably, family 19 chitinases were transferred from plants to bacteria by horizontal gene transfer [22] In plants, family 19 chitinases are thought to form part of a defense mechanism against chitin-containing fungal pathogens [36] The family 19 chitinases are thought to attack the hyphal tips, which are believed to consist of newly synthesized chitin that is not firmly crystallized [35] This is in accordance with the observation that family 19 chitinases generally have relatively low activities towards the more crystalline forms of chitin Only a few chitinolytic bacteria possess family 19 chitinases, and these also display antifungal activity [22,35] In bacteria, chitinases, primarily belonging to family 18, are usually thought to be produced for the exploitation of chitinous substrates as a source of nutrition Production of multiple enzymes with varying properties (endo-action or exo-action, processive or not, presence of additional substrate-binding domains, preference for soluble or insoluble substrates) is beneficial, because this enables the bacterium to use parallel and potentially synergistic strategies during chitin breakdown Chitin occurs in a variety of forms and copolymeric structures [37], which may require different chitinases for effective degradation It remains to be seen whether the two family 19 chitinases of S coelicolor simply add to the bacterium’s enzymatic repertoire 4896 for effective chitin turnover, or play a specific role in some form of interaction with fungi Experimental procedures DNA techniques The chiG gene was amplified from genomic DNA (ATCC BAA-471D) from S coelicolor A3(2) with: primer ChiGul_S.coeli-F, 5¢-GCATCGTCTCACATGGAGAAGTCC GACACCCGGA-3¢ (BsmBI restriction site is in bold type); and primer ChiG_S.coeli-R, 5¢-GCATGGTACCCTAAC AGCTCAGGTT-3¢ (KpnI restriction site is in bold type) PCR reactions were conducted with Phusion DNA polymerase (Finnzymes, Espoo, Finland) in a PTC-100 Programmable Thermal Cycler (MJ Research, Inc., Waltham, MA, USA) The amplification protocol consisted of an initial denaturation cycle of 30 s at 98 °C, followed by 30 cycles of 10 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C, followed by a final step of 10 at 72 °C Amplified fragments were ligated into vector pCRÒ4Blunt-TOPOÒZero Blunt TOPO (Invitrogen, Carlsbad, CA, USA) The gene fragments were excised from the TOPO vectors for insertion in an expression vector, using BsmBI and KpnI for cloning into NcoIKpnI-digested pETM11 vector (Gunter Stier, ă EMBL, Heidelberg, Germany) The pETM11 vector contains an N-terminal His6 tag followed by a TEV-protease cleavage site The final constructs were transformed into E coli BL21Star (DE) (Invitrogen) ChiG mutants (E68Q, E68A, E77Q and E77A) were made with the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), essentially as described by the manufacturer DNA sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer ⁄ Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3100 Genetic Analyser (Perkin Elmer ⁄ Applied Biosystems) Production and purification of recombinant protein One hundred and fifty milliliters of E coli BL21Star (DE3) transformants containing the pETM11–chiG construct were grown at 37 °C in LB medium with 100 lgỈmL)1 kanamycin at 225 r.p.m., to a cell density of 0.6 at 600 nm Isopropyl-b-d-thiogalactopyranoside was added to a final concentration of 0.4 mm, and the cells were further incubated for h at 30 °C, and harvested by centrifugation (9 820 g, at °C, Beckman Coulter Avanti J-25, Rotor JA14) The cell pellet was lysed by making a periplasmatic extract First, the cell pellet was resuspended in 15 mL of ice-cold spheroplast buffer (10 mL of m Tris ⁄ HCl, pH 8.0, 17.1 g of sucrose, 100 lL of 0.5 m EDTA, pH 8.0, and 200 lL of phenyl- FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS I A Hoell et al methanesulfonyl fluoride and incubated on ice for The cells were then harvested by centrifugation (7 741 g, at °C, Beckman Coulter Avanti JA25-5), the supernatant was removed, and the pellet was incubated for 10 at room temperature The pellet was then resuspended in 12.5 mL of ice-cold, sterile water, and incubated on ice for 45 s before supplementing with 625 lL of 20 mm MgCl2 After centrifugation (7741 g, at °C, Beckman Coulter Avanti JA25-5), the supernatant was pressed through a 0.20 lm sterile filter, supplied with 20 lL of 50 mm phenylmethanesulfonyl fluoride per 10 mL of extract, and stored at °C It has previously been shown that these extracts are good, relatively ‘clean’ starting points for purification of intracellularly produced chitinases [38] ChiG was purified on an Ni-NTA column (Qiagen, Venlo, The Netherlands) using a flow rate of mLỈmin)1 The column was equilibrated in 100 mm Tris ⁄ HCl buffer (pH 8.0), containing 20 mm imidazole After the protein sample was loaded, the column was washed with the starting buffer The His-tagged protein was then eluted with 100 mm Tris ⁄ HCl buffer (pH 8.0), containing 100 mm imidazole The purified protein was dialyzed against 20 mm Tris ⁄ HCl (pH 8.0) and stored at °C Removal of the (His)6 tag was preformed by mixing 0.1 mg of (His)6–ChiG with 75 lL of 10 · TEV-protease buffer (0.5 m Tris ⁄ HCl, pH 8.0, and mm EDTA), mm dithiothreitol, 0.005 mg of TEV-protease and dH2O up to 750 lL This mixture was incubated at 37 °C for h After incubation, the mixture was dialyzed against 100 mm Tris ⁄ HCl (pH 8.0) and 20 mm imidazole overnight The dialyzed mixture was then applied onto an Ni-NTA column, as described above The flow-through fraction, now containing the ChiG protein with no (His)6 tag, was dialysed against 20 mm Tris ⁄ HCl (pH 8.0) and stored at °C The protein produced via this procedure contains a threeresidue N-terminal extension (Gly-Ala-Met) compared to the mature wild-type enzyme Bacterial family 19 chitinase short (< 10 s) soak in mother liquor containing additional PEG400 to a final concentration of 15% Complete X-ray data were collected at beamline ID14EH3 at the ESRF in Grenoble, equipped with an ADSC Q4R detector Diffraction images were processed with mosflm [39] and scaled and merged with scala in CCP4 [40,41] The crystals belong to space group P21 with cell ˚ ˚ ˚ dimensions a ¼ 48.67 A, b ¼ 74.38 A, c ¼ 64.18 A and ˚ resolution b ¼ 108.6°, and diffracted to at least 1.5 A Crystal data and data collection statistics are summarized in Table Calculation of the Matthews coefficient suggested two molecules in the asymmetric unit The structure was solved by molecular replacement using cns [42] The search model was a polyalanine chain comprising residues 90–294 of the ChiC structure (1WVU) Two solutions in the cross-rotation function were readily identified, and a subsequent translation search gave the positions in the unit cell Side chains were progressively added, guided by information Table Crystal parameters, data collection and refinement statistics for Streptomyces coelicolor chitinase G (ChiG) Crystal parameters Crystal dimensions (mm) Space group Unit cell dimensions Data collection Source ⁄ beamline ˚ Wavelength (A) ˚ Resolution (A) u-range, Du (°) Temperature (K) Rmerge (%)a No of reflections Unique reflections Multiplicity Mean I ⁄ rI Refinement ˚ Resolution range (A) Structure determination and bioinformatics High-quality diffracting crystals of ChiG were obtained by the vapor diffusion method in hanging drops Prior to crystallization, ChiG was concentrated by using a Centricon Plus-20 Centrifugal Filter Device as described by the manufacturer (Millipore, Billerica, MA, USA) in 20 mm Tris ⁄ HCl (pH 8.0) to a final concentration of 10 mgỈmL)1 Equal volumes of the protein solution were mixed with the reservoir solution containing 13% (w ⁄ v) PEG8000 and 110 mm zinc acetate in 80 mm sodium cacodylate buffer (pH 6.5), and equilibrated against the reservoir solution at room temperature Crystals, in the shape of thin plates, grew to a final size of about 0.2 mm within a week Crystals were mounted in nylon loops and flash-frozen in liquid nitrogen following a Completeness (%)b Rwork (%) Rfree (%)c rms deviation from ideal geometry ˚ Bond lengths (A) Bond angles (°) Ramachandran distribution (%)d Most favorable regions Allowed regions Disallowed regions 0.2 · 0.2 · 0.05 P21 ˚ ˚ a ¼ 48.67 A, b ¼ 74.38 A, ˚ c ¼ 64.18 A, b ¼ 108.6° ESRF ⁄ ID14EH3 0.980 74.3–1.50 180, 1.0 100 7.6 (48.1) 250 071 (36 192) 68 373 (9865) 3.7 (3.7) 13.2 (3.0) 74.3–1.50 98.5 (97.3) 18.5 (24.3) 20.7 (26.9) 0.010 1.1 97.5 2.0 0.5 a Values in parentheses refer to the outermost shell of data (1.58– ˚ 1.50 A) b Values in parentheses refer to the outermost shell of ˚ data (1.54–1.50 A) c Five per cent of data, randomly distributed over the full resolution range, were flagged as belonging to the Rfree cross-validation set, not used in the refinement d According to COOT [43] FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4897 Bacterial family 19 chitinase I A Hoell et al from both the rA-weighted 2Fo–Fc and Fo–Fc maps, during several cycles of modeling using coot [43], following refinement with refmac5 [44] Water molecules were appended using the ‘add water’ function of coot Four peaks close to His67 ⁄ Glu182 and Asp184 in molecule A and His67 ⁄ Glu182 and Asp137 in chain B, originally modeled as waters, were replaced by zinc ions, based on the refined B-values and residual peak heights in the Fo–Fc map These zinc ions originate from the crystallization buffer The main chain was readily traced from residue Phe12 all the way to the C-terminal Cys215 A few side chains at the protein surface are flexible, with no distinct conformation Refinement of ChiG with zero occupancy for residues 170–183 confirmed the (slightly) different conformation for ChiG in the 178–183 region, as judged by inspection of the difference Fourier electron density map The final model comprises 408 residues in two chains, four zinc ions and 324 water molecules Coordinates and structure factors have been deposited in the Protein Data Bank, accession code 2CJL To create the alignment of Fig 1, structural superposition with other family 19 chitinases was performed using the protein structure comparison service SSM at the European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/ssm) [45] In cases where analysis of the anomeric configuration of the newly formed degradation products was desirable, reactions were performed with higher enzyme concentrations (20 nm) and very short incubation times (approximately 15 s) To stabilize the anomeric ratio as fast as possible and to avoid reaching the anomeric equilibrium, reactions were stopped by freezing on liquid nitrogen and samples were stored at ) 80 °C until analyzed Ten microliter samples of the reaction mixtures were injected with a Gilson 234 autoinjector immediately after thawing (that is, samples were not ‘stored’ in the autoinjector) Analyses of the degradation of a-chitin and b-chitin were conducted by incubating 100 lL solutions containing mgỈmL)1 of b-chitin (squid pen b-chitin, lm in size; Seikagaku, Tokyo, Japan) or mgỈmL)1 a-chitin (crab-shell a-chitin, Sigma) and 20 nm purified ChiG in 50 mm sodium acetate buffer (pH 6.0) at 37 °C and 230 r.p.m for periods varying from (just after addition of enzyme) to 24 h Reactions were stopped by adding one volume of 70% (v ⁄ v) acetonitrile, and samples were stored at ) 80 °C until they were injected with a 234 autoinjector (Gilson) Activity tests with a colloidal chitin substrate, CM-chitin RBV (LOEWE Biochemica GmbH, Munchen), and with ă 4-MU-(GlcNAc)2 (Sigma) or 4-MU-(GlcNAc)3 (Sigma) were performed as described earlier [46] Enzymology Acknowledgements Protein concentrations were determined according to Bradford with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA) with BSA as a standard Analyses of the specific activity against chitooligosaccharides were performed in 100 lL reaction mixtures containing 200 lm (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, or (GlcNAc)6 (Sigma, St Louis, MO, USA), 0.1 mgỈmL)1 BSA and 0.25 nm purified ChiG in 50 mm sodium acetate buffer (pH 6.0) In the case of the (GlcNAc)3 substrate, the enzyme concentration was 12.5 nm In the case of ChiG mutants, the enzyme concentration was varied between 250 nm and 500 nm (see below for details) All the reaction mixtures were incubated at 37 °C for several hours, with regular sampling Sixty microliter samples of the reaction mixture were transferred to new tubes containing 60 lL of 70% acetonitrile, to stop the reaction, and stored at ) 20 °C until they were analyzed by HPLC at room temperature All reactions were analyzed in triplicate HPLC analysis of 20 lL portions of the stored reaction mixtures was performed on a Gilson HPLC System (Gilson, Inc., Middleton, WI, USA), equipped with a Tosoh TSK-Gel amide-80 column (0.46 internal diameter · 25 cm) (Tosoh Bioscience, Montgomeryville, PA, USA), and a 234 autoinjector (Gilson) The liquid phase consisted of 70% (v ⁄ v) acetonitrile, the flow 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G, Horn SJ & Eijsink VGH (2005) Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1 Biochim Biophys Acta 1748, 180–190 DeLano WL (2004) The Pymol Molecular Graphic System San Carlos, CA Supplementary material The following supplementary material is available online: Fig S1 SDS ⁄ PAGE analysis of purified chitinase G (ChiG) Fig S2 Degradation of b-chitin Fig S3 Close-up view of the rA-weighted 2Fo–Fc electron density map in a representative part of the structure of chitinase G (ChiG) This material is available as part of the online article from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS ... were made with PYMOL [47] able sequences, some bacterial family 19 chitinases have catalytic domains that are at least as large as those of the plant enzymes and that may contain at least six... 35 Kawase T, Yokokawa S, Saito A, Fujii T, Nikaidou N, Miyashita K & Watanabe T (2006) Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S coelicolor A3 (2)... Haemophilus influenzae, and Pseudomonas aeruginosa Although many plant family 19 chitinases are known, crystal structures are available for only two of these [9,15–17] The first structure of a bacterial