Differential recognition of animal type ß4-galactosylated and {alpha}3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

Harry Boer1,4, Nana Munck4, Jari Natunen2,5, Gerd Wohlfahrt3,4, Hans Söderlund4, Ossi Renkonen5 and Anu Koivula4

4 VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland, and 5 University of Helsinki, Helsinki, Finland

Received on June 2, 2004; accepted on June 28, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these ({alpha}/ß)8-barrel fold enzymes to recognize ß-1,4-galactosylated and {alpha}-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (–2–1–+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites –4 and –3 of Chit42, and at the subsite –4 of Chit33. Fucose units can be accepted as a branch at the putative –3 and –4 sites of Chit33 and as a branch point at –3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.

Key words: ({alpha}/ß)8-barrel fold / chitinases / chito-oligosaccharides / molecular modeling / substrate specificity


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chitinases (EC 3.2.1.14) hydrolyze chitin, a linear polymer of N-acetylglucosamine residues, and produce soluble chito-oligosaccharides. The catalytic domains of chitinases belong to the glycosyl hydrolase families 18 and 19 (http://afmb.cnrs-mrs.fr/cazy/gh.html). Chitinases are found in various organisms, for example, bacteria, plants, and fungi, and recently chitinase-type proteins belonging to family 18 have also been identified in humans (Boot et al., 1998Go, 2001Go; Fussetti et al., 2002Go). One of them is a reliable marker for a genetic lysosomal storage disorder, and another seems to be an inactivated, chitinase-like protein. Other noncatalytic, possibly lectin-like proteins in family 18 are seed storage proteins, concanavalin B, and narbonin (Fussetti et al., 2003Go; Hennig et al., 1992Go, 1995Go; Houston et al., 2003Go). Family 18 also contains nonchitinolytic enzymes, such as endo-ß-N-acetylglucosaminidases F1 and H, which are involved in the processing of high-mannose asparagine-linked oligosaccharides on glycoproteins (Rao et al., 1995Go; Van Roey et al., 1994Go). All family 18 proteins have an ({alpha}/ß)8-barrel fold, where the substrate binding cleft is formed by loops positioned between the carboxyl-terminal end of the ß-strands and the amino-terminal end of the helices (Henrissat and Davies, 1997Go).

Substrate binding as well as the catalytic mechanism have been studied for a rubber tree chitinase, hevamine, and for the two Serratia marcescens chitinases A and B. The 3D structures of these enzymes have also been solved, revealing that hevamine has an open active site architecture, while both bacterial chitinases have a more closed active site. The 3D structure of a chitinase from a pathogenic fungus, Coccioides immitis, in complex with allosamidin has also been solved recently (Bortone et al., 2002Go; Hollis et al., 2000Go). In all cases the extended substrate-binding site allows binding of several NAG (N-acetylglucosamine, N-acetyl-ß-D-glucosamine) units (Brameld et al., 1998Go; Papanikolau et al., 2001Go; Terwisscha van Scheltinga et al., 1994Go, 1995Go; van Aalten et al., 2001Go).

Carbohydrate sequences on glycoproteins, glycolipids, and proteoglycans are key ligands in different molecular recognition systems. In particular, cell-adhesion and cell-activation events triggered by carbohydrate–protein interaction are among the current topics of active research. The lactosamine Galß1-4GlcNAc and its fucosylated variant Lewis x, that is, Galß1-4(Fuc{alpha}3)GlcNAc, are animal-type carbohydrates, which are important in human and mammalian biology. N-acetyllactosamine motifs function as ligands for the galectin family of lectins (Hughes, 1997Go), while the fucosylated variants serve as selectin ligands (MacEver et al., 1995Go; Varki, 1994Go). Development of specific proteins for cleaving, synthesis, or binding analysis of those oligosaccharide structures would provide useful tools for glycobiology studies, diagnostics, and even development of glyco drugs regulating carbohydrate recognition.

We are interested in the substrate-binding specificity of glycosyl hydrolase family 18 chitinases and whether it could be engineered toward medically important oligosaccharides. We report here the purification of two fungal chitinases, Trichoderma harzianum Chit33 and Chit42, of family 18 and their enzymatic characterization using a panel of different soluble chitinous substrates and inhibitors. These two chitinases were chosen based on the fact that the smaller 33-kDa chitinase (Chit33) is homologous to the plant chitinase hevamine and is very likely to share structural similarities with this and other small plant chitinases, and the 42-kDa enzyme (Chit42) shows homology to bacterial and other fungal chitinases. We used homology modeling to build 3D structures for both fungal chitinases in complex with chito-oligosaccharides, based on the published crystal structures of family 18 enzymes. The enzyme complex structures were used to analyze the degradation patterns and establish binding models for different substrates. We are particularly interested in the specificity of the two Trichoderma chitinases toward animal-like ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides. This work forms a basis for understanding and modifying the substrate-binding specificity of one or both enzymes toward animal type of oligosaccharides.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification and characterization of the two fungal chitinases
Both T. harzianum chitinases were purified from the fungal culture supernatant using an anion exchange column followed by a gel filtration. When analyzed by Coomassie blue–stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), a single band with the expected molecular weight of 33 and 42 kDa, respectively, was observed after purification. The N-terminal sequence analysis revealed in both cases the expected amino acid sequences, verifying correct processing of the secreted products.

Substrate specificity and kinetic parameters
The pH optima of both chitinases were measured at 30°C, demonstrating that the purified Chit33 has an optimal activity around pH 5 (in 50 mM NaAc buffer) on NAG3-p-nitrophenyl (PNP), and Chit42 at pH 6 (in 25 mM KPi buffer) on NAG2-4-methylumbelliferyl (MU) (data not shown). These optimum pH values were used in the all the kinetic measurements reported here. First, different-length chromophoric chito-ologosaccharides were used to characterize the degradation patterns of the two enzymes. Neither one of the chitinases showed activity against NAG-MU. Both NAG2 derivatives (NAG2-MU and NAG2-PNP) were very poor substrates for Chit33, and the enzyme seems to have a preference for longer chito-oligosaccharides (Table I). Chit42, on the other hand, is able to degrade both the NAG2-MU and NAG2-PNP efficiently. The trisaccharide derivative, NAG3-PNP, is a good substrate for both chitinases. High-performance liquid chromatography (HPLC) analysis of the hydrolysis products showed that Chit33 cleaves this substrate only at one position resulting in the formation of PNP and NAG3, whereas Chit42 cleaves NAG3-PNP at two different positions leading to the formation of NAG2, NAG-PNP, NAG3 and PNP (Table I). NAG3-MU is also degraded by both enzymes, but poor solubility of the substrate makes a detailed kinetic analysis difficult (data not shown).


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Table I. Kinetics and degradation of chromophoric and fluorogenic chito-oligosaccharides

 
More detailed analysis showed that the cleavage of the chromophoric group of NAG3-PNP by Chit33 and NAG2-PNP by Chit42 follows Michaelis-Menten type of kinetics and thus allowed the measurement of Km and kcat values (listed in Table I). When NAG2-MU was used at higher substrate concentrations (>250 µM) with Chit42, the reaction was inhibited. Interestingly, similar substrate inhibition has also been observed for the homologous bacterial and fungal chitinases (Brurberg et al., 1996Go; Fukamizo et al., 2001Go).

The reactivity of both enzymes for a series of galactose- and glucose-containing MU-oligosides was also determined (Table I). No hydrolysis of non-NAG-containing substrates Gal-MU, Glc-Gal-MU, and Glc2-MU was observed for either Chit33 or Chit42 (Table I).

Inhibition by transition state analogs and chito-oligosaccharides
Both enzymes were further investigated using a series of different inhibitors (Figure 1). The first inhibitor, allosamidin, is a natural pseudotrisaccharide produced by some Streptomyces species, and it is known to be a potent inhibitor of other family 18 chitinases. Additionally, two synthetic inhibitors were used, which are N-phenylcarbamate derivatives of either di-N-acetyl-chitobiose, or of N-acetyl-ß-D-glucosamine. Both inhibitors have the modification at the position C1 of the pyranose ring and they were designed to mimick a transition state of a distorted carbohydrate ring (Beer et al., 1990Go). The results from the inhibition studies are shown in Figure 1, where the ratio of the rate in the presence (v) and absence (v0) of the inhibitor is plotted as a function of the inhibitor concentration (Ley et al., 1997Go). All experiments in one panel are performed at the same enzyme and substrate concentration, and the initial rates were measured by taking samples of the reaction mixture at four different time points. Allosamidin shows the strongest inhibitory effect both on Chit33 and Chit42, and Chit42 seems to be more strongly inhibited by allosamidin as compared to Chit33 (Figure 1). From Figure 1 it can also be seen that the two N-phenylcarbamate derivatives do not inhibit Chit33 under the experimental condition. In the case of Chit42, a small inhibitory effect is observed for the di-N-acetyl-chitobiose derivative, but the inhibition is not much stronger than with di-N-acetyl-chitobiose, the product (sugar) of this enzyme. NAG3 and NAG4, which are not cleaved by Chit33, also inhibit the enzyme, and the inhibitory effect is strongest for the longer chito-oligosaccharide NAG4.



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Fig. 1. Inhibition of Chit33 (A) and Chit43 (B) by transition state analogs and chito-oligosaccharides. The ratio of the rate in the presence (v) and absence (v0) of inhibitor is plotted as a function of the inhibitor concentration. All experiments in one panel were performed at the same enzyme and substrate concentration, and the initial rates were measured by taking samples at four different time points. 200 µM PNP-NAG3 (Chit33) and 200 µM MeUmb-NAG2 (Chit42) were used as substrates. The inhibitors that were tested are: Allosamidin (diamonds), N-phenylcarbamate derivatives of di-N-acetyl-chitobiose: O-(chitobiosylidene) amino N-phenylcarbamate (x) and N-acetyl-ß-D-glucosamine: O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (+), NAG2 (circles), NAG3 (upside-down triangles) and NAG4 (triangles).

 
MS analysis of hydrolysis reactions catalyzed by purified T. harzianum chitinases with native and modified animal-type ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides
As described in the Materials and methods section, the mass spectroscopic (MS) quantification of medium sized chito-oligoaccharides was first verified with binary model mixtures. For the quantification of the hydrolysis products, the enzyme was denatured and removed from the reaction mixture by ion-exchange chromatography. This MS analysis method has also been used earlier in saccharide quantifications (Natunen et al., 2001Go; Räbinä et al., 1997Go), and it is a very useful and fast method for degradation pattern analysis. The semi-quantitative or quantitative data about NAG3–NAG6-size chito-oligoaccharides (see the Materials and methods) give a reasonable amount of information about the cleavage mechanism of the enzymes. The presence of NAG2 in the spectra indicates that chitobiose is released at least in part of the hydrolysis reactions but leaves open the question whether two NAG units besides NAG2 could also occasionally be cleaved from the reducing end of an oligosaccharide. The possible release of single NAG units would not, however, change the results about the subsites acceptable for the modification closest to the cleavage position. For the following part of the discussion, it should be kept in mind that NAG2 refers also to the possibility of some amounts of NAG.

Figure 2 shows the different products formed from the cleavage of unmodified, galactosylated, and fucosylated NAG6 (hexa-N-acetyl-chitohexaose) by Chit33 after three different incubation times (see figure legend for details). The degradation products from the hydrolysis of NAG6 by Chit33 are shown in Figure 2 (top). NAG4 and NAG2 were the major cleavage products, and a small amount of NAG3 was also observed in all three hydrolysis experiments. NAG4 was rather resistant toward further hydrolysis by Chit33, even under the most harsh reaction conditions. In addition to the cleavage products, transglycosylation was occurring, as NAG7 was also detectable in all reaction mixtures. The second substrate, Gal-NAG6 (ß-1,4-galactosylated hexa-N-acetyl-chitohexaose), was cleaved mainly into Gal-NAG3 (and NAG3), but also some minor amounts of Gal-NAG4 could be seen (Figure 2, middle). Interestingly, Gal-NAG4 seems to be effectively cleaved further into Gal-NAG3 and NAG units by Chit33 (Figure 2, middle). Transglycosylation leading to longer oligosaccharide formation was also occurring because Gal2-NAG6 and Gal-NAG7 could be detected (Figure 2, middle). The third substrate, {alpha}-1,3-fucosylated hexa-N-acetyl-chitohexaose, Fuc-NAG6 (NAGß1-4(Fuc{alpha}1-3)NAGß1-4NAG4), was cleaved effectively into Fuc-NAG5 and NAG units (Figure 2, botom). The digest contained also lots of Fuc-NAG4, but very little of NAG2, implying that the Fuc-NAG4 is a cleavage product from Fuc-NAG5. Fuc-NAG4 appeared rather stable against further hydrolysis by Chit33.



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Fig. 2. The end product spectrum from the hydrolysis of ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides by T. harzianum Chit33. The degradation products produced from the modified chito-oligosaccharides by Chit33 under three different conditions (see legend) were measured using MALDI-TOF MS as described in the Materials and methods section. The quantities of the oligosaccharide products from the hydrolysis of different oligosaccharides under each three condition are shown as molar percentages from the heights of the MS peaks. The accuracy in quantification is good for trisaccharides and longer oligosaccharides, whereas the data for NAG and NAG2 is more semi-quantitative. The reaction mixtures contained 6 nmol of the substrate in 10 µl 50 mM NaAc, pH 5.0. Substrates: NAG6 (top panel), Galß1-4NAG6 (middle panel), and NAGß1-4(Fuc{alpha}1-3)NAGß1-4NAG4 (lower panel).

 
Figure 3 shows the results of the hydrolysis of the same three compounds by Chit42. The primary cleavage of NAG6 by Chit42 produces mainly NAG2 and NAG4, but also two molecules of NAG3 are produced frequently (Figure 3, top). The overall cleavage reaction of NAG6 by Chit42 seems to be faster than by Chit33. Besides the hydrolysis, transglycosylation is also observed with Chit42 on NAG6. Transglycosylation yielding NAG7 would seem to be more frequent than the reaction giving NAG8 (Figure 3, top). The primary cleavage of Gal-NAG6 by Chit42 releases mainly Gal-NAG4 and NAG2 and also some Gal-NAG3 and NAG3. In a secondary cleavage reaction, Gal-NAG4 is converted into Gal-NAG2 and NAG2. Also here some transglycosylation was observed, with Gal-NAG7 and di-Gal oligosaccharides Gal2-NAG6 and Gal2-NAG7 as the reaction products. In the third hydrolysis reaction, Fuc-NAG6, was used as the substrate (Figure 3, bottom). The primary cleavage by Chit42 gives mostly Fuc-NAG4 and NAG2, but Fuc-NAG5 and NAG are also formed. Fuc-NAG4 does not seem to be degraded by Chit42, and Fuc-NAG2- and Fuc-NAG3-releasing reactions do not occur.



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Fig. 3. The end product spectrum from the hydrolysis of ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides by T. harzianum Chit42. The degradation products produced from the modified chito-oligosaccharides by Chit42 using different incubation times (see legend) were measured using MALDI-TOF MS as described in the Materials and methods section. The quantities of the oligosaccharide products from the hydrolysis of different oligosaccharides under each condition are shown as molar percentages from the heights of the MS peaks. The accuracy in quantification is good for trisaccharides and longer oligosaccharides, whereas the data for NAG and NAG2 is more semi-quantitative. The reaction mixtures contained 6 nmol of the substrate in 10 µl 50 mM KPi, pH 6.0. Substrates: NAG6 (top panel), Galß1-4NAG6 (middle panel), and NAGß1-4(Fuc{alpha}1-3)NAGß1-4NAG4 (lower panel).

 
Homology models of Chit33 and Chit42
Both T. harzianum chitinases Chit33 and Chit42 are single-domain proteins lacking the ~ 120-amino-acids-long chitin-binding domain, which is found, for example, in S. marcescens chitinase A. Chit42 shares an overall sequence identity of 53% with the template CiX1; especially in the ligand-binding groove there are very few conservative exchanges (Figure 4). The stereochemical quality of the model structure according to PROCHECK (Laskowski et al., 1993Go) is very good. In both the template and the model, three residues are outside allowed regions of the Ramachandran plot. Chit33 shares only a moderate overall homology with hevamine (31%), but more than 70% of the binding site residues are conserved (Figure 4). Because of this low homology, the stereochemical quality (Laskowski et al., 1993Go) of the Chit33 model structure is less good compared with the Chit42 model. In the Chit33 model 8 residues more than in the template are found outside allowed regions of the Ramachandran plot. The differences between the chitinase models and the template structures are mainly found in loop areas, although in Chit42 also most of the loops are conserved. In our Chit33 model two putative disulfide bridges are found, and they are also conserved in the hevamine template (which has altogether three disulfide bridges). In the C. immitis CiX1 structure and our Chit42 model, no disulfide bridges are found. As described by Hollis et al. (2000)Go, S. marcescens chitinase A has several insertions when compared with C. immitis CiX1 and T. harzianum Chit42, but the ({alpha}/ß)8 core structure is well conserved and there is a high degree of conservation in the substrate-binding groove of these chitinases. Cis-peptide bonds seem to be an important factor for the binding site conformation of chitinases. Four can be found in C. immitis CiX1 (Hollis et al., 2000Go), and they are likely to be conserved in the Chit42 structure.



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Fig. 4. Structural models of the substrate-binding sites of T. harzianum Chit33 and Chit42. The chito-oligosaccharides are presented in green. Only the side chains of residues, which form the substrate-binding sites are shown and labeled. (Left) Chit33 in complex with chitohexaose. Amino acid differences between Chit33 (white) and hevamine (blue) are indicated. The subsites are designated as –4, –3, –2, –1, +1, and +2, where –1/+1 defines the cleavage point (Henrissat et al., 1998Go). (Right) The substrate-binding site of Chit42 in complex with chitoheptaose. Amino acid differences between Chit42 (white), chitinase A (blue), and Ci1X (red) are indicated. The subsites are designated as –5, –4, –3, –2, –1, +1, and +2.

 
Complex structure models
Superimposing the Chit42 model with the crystal structure of S. marcescens chitinase A complexed with octaacetylchito-octaose and the Chit33 model with the hevamine structure complexed with allosamidin and tri-N-acetyl-chitotriose suggests that the substrate-binding site of both T. harzianum chitinases is ~ 35 Å long (Papanikolau et al., 2001Go; Terwisscha van Scheltinga et al., 1996Go). However, the overall shape of the substrate-binding sites differs in the two T. harzianum chitinase models. T. harzianum Chit42, similarly to the C. immitis CiX1 and S. marcescens chitinase A, seems to have a much deeper groove for substrate-binding than Chit33. This is due to an additional ß-sheet domain in the Chit42 model structure, which is involved in binding site formation.

Figure 4 shows models for binding of chito-oligosaccharide chains to T. harzianum Chit33 and Chit42, respectively. Due to conservation of most of the amino acids in the binding site, the twisted conformation of the NAG8 chain, observed in the crystal structure of S. marcescens chitinase A, also fits well into the binding site of the Chit42 model (Figure 4). Chit33 (like hevamine) seems to contain at least six and Chit42 (similar to S. marcescens chitinase A) at least seven subsites for NAG units. The enzyme–substrate complex models show that the binding groove of Chit42 contains more aromatic residues interacting with the substrate as compared to Chit33. There are five tryptophans, seven tyrosines, and one phenylalanine in the binding site of Chit42, whereas there are only one tryptophan, two tyrosines, and one phenylalanine in Chit33 (Figure 4). In Chit42 the aromatic residues are spread along the binding groove and are major determinants for formation of the subsites. In Chit33 the aromatic residues are located near the catalytic residues around subsite –1, either stacking with the oligosaccharide or stabilising the correct structure of the binding pocket.

In our Chit42 complex structure model, the hydrophobic face of the pyranose ring at the –1 site stacks with the Trp379 side chain, and Tyr44 and Phe72 form the wall in that part of the binding cleft. The residues Tyr240, Tyr44, Phe72, and Trp379 of Chit42 (corresponding to Tyr202, Tyr10, Phe41, and Trp279 of Chit33) are located in the immediate proximity of the putative catalytic amino acids and are conserved in all chitinases discussed in this paper (see Figure 4). In the subsites –3 and –2 of Chit42, most residues (Glu317, Arg53, Thr133, Glu380, Phe72, and Trp48) are conserved and are also found in the nearest homologs, chitinase A and CiX1. However, these residues are not conserved in Trichoderma Chit33 and hevamine. The sugar residue bound at the subsite –5 of Chit42 stacks against a conserved Tyr51 (Tyr170 in chitinase A and Tyr50 in CiX1), and a conserved Trp 316 flanks subsite –4 (Trp472 in chitinase A and Trp315 in CiX1). In Chit33, Ala55, Asn14, Gln13, and Asn43 participate in the formation of the binding cleft at the subsites –4 and –3 (Figure 4). Subsites +1 and +2 are lined by polar residues, Asn203, Asn204, and Gln177, found also in hevamine. Near the site where the glycosidic bond is cleaved, on one side of the binding cleft there is a very flexible loop (found both in Chit33 and hevamine), which extends from the –1 site to the +2 site and includes residues Gly249 to Gly253 of Chit33.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The ({alpha}/ß)8 topology is found in several families of glycosyl hydrolases with very different substrate specificity and a very low overall sequence homology (Henrissat and Davies, 1997Go; Nagano et al., 2001Go). The catalytic residues have been identified in a number of these enzymes, and the presence of conserved unusual nonproline cis-peptide bonds provides further evidence for an evolutionary relation between ({alpha}/ß)8, or TIM-barrel, glycosyl hydrolases (Herzberg and Moult, 1991Go; Stewart et al., 1990Go). Besides glycosyl hydrolases, the TIM-barrel fold is also found in other enzyme classes, demonstrating its versatile functions (Orengo et al., 1994Go). It has been speculated that artificial enzymes could be designed and engineered on the basis of this fold, and recently it was demonstrated that the ({alpha}/ß)8 barrel fold is indeed a good scaffold for changing the substrate specificity of a synthase into isomerase by directed evolution (Altamirano et al., 2000Go). The two T. harzianum chitinases studied here both belong to the glycosyl hydrolase family 18 and have an ({alpha}/ß)8 barrel fold. The extended binding sites of family 18 chitinases have been reported to accommodate up to six or seven sugar moieties. The binding site cleft of chitinases provides specific interactions to the sugar units through a limited number of protein loops. Thus the amino acid residues that interact with the substrate are not distributed over the whole sequence, as observed in some other carbohydrate active enzymes (e.g., cellulases and xylanases). These factors make the loops suitable targets for random mutagenesis approaches to change the binding specificity of chitinases.

T. harzianum chitinases have been mostly studied because of their potential in biocontrol against other fungal pathogens and nematodes causing diseases in agricultural crops (de la Cruz et al., 1992Go; Hayes et al., 1994Go; Limón et al., 1995Go). The suggested action of chitinases there is to hydrolyze the chitin containing cell walls of the plant pathogens. We report here the enzymatic characterization of the two major chitinases of Trichoderma, Chi42 and Chi33, on various small oligosaccharides. We were particularly interested in their action on synthetic, nonnatural substrates containing galactose or fucose units. Interestingly, however, there are now reports that, for example, the fucosylated chito-oligosaccharide structure also mimics the structure of a reported nod-factor (Olsthoorn et al., 1998Go). Degradation of such structures by a fungal or plant (chitinase-type) enzyme could thus be reactions found also in nature (Ovtsyna et al., 2000Go).

The reasonably high homology particularly of the T. harzianum Chit 42, but also of the Chit33, with the solved 3D structures of other family 18 chitinases should allow reliable model building, especially for the ligand-binding sites in which we are interested here. The modeled structures of the two T. harzianum chitinases differ in the depth of the substrate-binding site, Chit42 having a deeper groove for chito-oligosaccharide binding. We suggest that the extra ß-sheet domain in the Chit42 model and a different tryptophan and tyrosine distribution in the substrate-binding sites are likely to be important factors that explain the different kinetic behaviour of these two enzymes in degradation studies on various chito-oligosaccharides. Figure 5 shows the subsite binding models of Chit33 and Chit42 on galactosylated and fucosylated chito-oligosaccharides based on our degradation pattern analysis (Figures 2 and 3) and the structural models (Figure 4). As can be seen from Figure 5, we suggest that Chit33 has at least six subsites (–4 -> +2) and Chit42 at least seven subsites (–5 -> +2). Both enzymes cleave preferentially between the second and third sugars from the reducing end of the substrate. Our kinetic data further suggest that Chit42 can degrade chitohexaose (NAG6) in a processive manner into (three) NAG2 units, and Chit33 produces NAG4 and NAG2, which are not further degraded. Apparently both enzymes have (at least) two different binding modes for chitohexaose. In the preferred binding mode NAG6 occupies the subsites –4 -> +2, but the chain can also bind further beyond the cleavage point, that is, to subsites –3 ->+3, leading to formation of two NAG3 units. A similar cleavage pattern has been observed in our earlier studies on the cellohexaose hydrolysis by Trichoderma reesei cellobiohydrolase, Cel6A (Harjunpää et al., 1996Go).



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Fig. 5. Schematic representation of the different subsite binding modes of ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides by T. harzianum Chit33 and Chit42. Chit 42 has seven (–5 -> +2) and Chit33 has six subsites (–4 -> +2), where the nonreducing end of the chain binds to – subsites and reducing end to + subsites (Henrissat et al., 1998Go). NAG units present in the product side (at + subsites) are not shown.

 
The family 18 chitinases are supposed to cleave the glycosidic linkage of the chito-oligosaccahride (between subsites +1 and –1) with retention of the configuration. It has also been suggested that chitinases apply a substrate-assisted catalysis mechanism involving the catalytic acid and the carbonyl oxygen atom of the substrate's N-acetyl group at subsite –1 as the nucleophile (Brameld et al., 1998Go; Terwisscha van Scheltinga et al., 1995Go; Tews et al., 1997Go; van Aalten et al., 2001Go). According to this hydrolysis mechanism, NAG is always needed to occupy the –1 site for the catalysis to occur. Our hydrolysis results with different MeUmb-saccharides support this reaction mechanism for both Trichoderma chitinases, because only a NAG unit was accepted at subsite –1 (Table I). In addition, allosamidin was the most potent inhibitor for both chitinases, which further supports the substrate-assisted catalysis mechanism. Allosamidin binds, according to the published family 18 chitinase complex structures, to subsites –3, –2, and –1 (Terwisscha van Scheltinga et al., 1995Go; van Aalten et al., 2001Go). The two synthetic N-phenylcarbamate derivatives tested appear either not to mimic the transition state sufficiently and/or to possess too short saccharide chains to be effective inhibitors under the experimental conditions used here. We also observed that both chitinases can catalyze transglycosylation reactions of the various oligosaccharides (Figures 2 and 3, middle panels) similar to other retaining hydrolases (Harjunpää et al., 1999Go; Sasaki et al., 2002Go). This led, for example, to the formation of products containing two galactose units when ß-1,4-galactosylated hexa-N-acetyl-chitohexaose was used as a substrate for Chit42 (Figures 2 and 3, middle panel). Taking the active site configuration in consideration, it is not implausible that a ß-1,4 bond between a galactose and a NAG unit is formed. These types of enzymatic transglycosylation reactions are of interest because the chemical synthesis of complex oligosaccharides is usually very tedious (Aguilera et al., 2003Go).

As seen from Figure 3 (middle) Chit42 can degrade animal type Gal-NAG6 (ß-1,4-galactosylated hexa-N-acetyl-chitohexaose) into Gal-NAG4 and NAG2 (or two NAG units) or into Gal-NAG3 and NAG3 units. Gal-NAG4 can also be further cleaved into Gal-NAG2 and NAG2, which are the main products after extensive hydrolysis. These data imply that Gal units can be accommodated in both subsites –4 and –3 of Chit42 (Figure 5). Chit33, on the other hand, releases from the nonreducing end of Gal-NAG6 both Gal-NAG4 and Gal-NAG3 but not Gal-NAG2 (Figure 2, middle). This implies that Gal can be accommodated only at the subsite –4 of Chit33 (Figure 5). The structural model of Chit42 reveals a conserved Thr133 residue located at subsite –3, which in the solved homologous structures is shown capable of making a hydrogen bond with the N-acetyl group of NAG (Bortone et al., 2002Go; Papanikolau et al., 2001Go). Another conserved hydrogen bond donor, Glu317 (in Chit42), can form an additional hydrogen bond with the hydroxyl group of NAG (at position C6). On the other hand, Trp134 aligning subsite –3 stacks with the NAG ring structure but could in principle also stack against a Gal ring.

Galactosyl differs from a NAG moiety in that the hydroxyl group at C4 has an axial configuration, and it is missing the N-acetyl group at C2, thus making it smaller. Thus, on the basis of our structural model, it is plausible that a smaller Gal unit can be accommodated at subsite –3 of Chit42. At the end of the groove, at subsites –5 and –4 of Chit42, there is overall more space, and tight interactions between the protein and the saccharide units are not very likely. This probably allows more tolerance toward different substrates so that a galactose unit could be accepted (instead of a NAG unit) at both of these subsites. In the Chit33 model, subsites –3 and –4 are located on the surface of the enzyme and are more exposed when compared with the corresponding subsites in Chit42. The conserved Gln13 at subsite –3 is capable of forming a hydrogen bond with the N-acetyl group of NAG unit in subsite –3, and at subsite –4 (of Chit33), which tolerates a galactose, mainly interactions with the protein backbone are observed. Contrary to Chit42, no aromatic residues are found in Chit33 in these two subsites –3 and –4. No major contribution is expected from the axial 4-hydroxyl group in galactose, because it is proposed to point out of the binding sites in both chitinases.

The main end products of Fuc-NAG6 ({alpha}-1,3-fucosylated hexa-N-acetyl-chitohexaose) hydrolysis by Chit42 are Fuc-NAG4 and NAG2 (or two NAG units) (Figure 3, bottom). In the case of Chit33, both Fuc-NAG5 and Fuc-NAG4 were produced from the nonreducing chain end (Figure 2, bottom). Fuc-NAG4 appears to be a rather stable product with both chitinases. The end product Fuc-NAG5 occupies the subsites –5 -> –1 of Chit33, in such a way that the fucose unit is at the branch point to the NAG unit at subsite –4 (Figure 5). In the case of the shorter end product Fuc-NAG4, the fucose unit is at the branch point to the NAG unit bound to the subsite –3 in both enzymes. The conformation of an {alpha}-1,3-fucosylated saccharide has been determined in solution by nuclear magnetic resonance (NMR), showing that the branching fucose ring stacks against a NAG unit in the linear chain. It is plausible that also in the bound form the fucose unit stacks against a NAG unit occupying the subsite –3 (or –4) (Räbinä et al., 1997Go).

Overall, our hydrolysis results with modified chito-oligosaccharides show that the most significant enzyme–substrate contacts are localized at subsites –1, +1, and +2 and that they are mediated by aromatic and charged residues. Our results give experimental evidence that less specific sugar interactions are found at subsites –5 -> –2 of Chit42 and in –4 -> –2 of Chit33, as these subsites tolerate flexibility in sugar composition. This type of flexibility in recognition should allow a good starting point for future directed evolution and protein-engineering experiments aiming at altered substrate specificity. Furthermore, the degradation pattern analysis of the chito-ologosaccharides gives some ideas for design of modified chromophoric chitinase substrates for screening purposes. We have also demonstrated that wild-type Chit42 has affinity toward a saccharide affinity column containing immobilized NAG3, whereas Chit33 elutes out already in the flow-through (data not shown). Although these data give further support to the notion that Chit42 has stronger affinity toward short chito-oligosaccharides than Chit33, they also demonstrate that this type of sugar column could be used to screen for altered substrate specificity of mutated chitinases.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Growth of the Trichoderma strains expressing Chit33 and Chit42
The T. harzianum P1 endochitinase of 42 kDa, Chit42, was purified from the earlier constructed T. reesei strain VTT-D-95434 expressing the prepro form of the chitinase gene under a strong cbh7A promoter (Margolles-Clark et al., 1996Go). A 50 ml preculture (2% whey, 1% distillers spent grain, 1.5% KH2PO4, 0.5% (NH4)2SO4, 0.6 g/L MgSO4.7H2O, 0.6 g/L CaCl2.2H2O, 5 mg/L FeSO4.7H2O, 1.6 mg/L MnSO4.H2O, 1.4 mg/L ZnSO4.7H2O, and 3.7 mg/L CoCl2.6H2O) was inoculated with the spore suspension and grown at 28°C. The enzyme production was checked every 24 h using Coomassie-stained SDS–PAGE gels. After 72 h, the preculture was used to inoculate a 2-L culture, which was grown for 72 h at 28°C. The culture supernatant was harvested by filtering the culture over a Whatman GF/B filter and stored at –20°C.

The 33-kDa chitinase Chit33 was produced in T. harzianum strain T30 as described by Limón et al. (1995)Go. In this strain the Chit33 can be selectively produced on minimal medium containing glucose. A 50-ml Trichoderma minimal medium + 0.2% proteose peptone preculture (2% glucose, 0.2% proteose peptone, 1.5% KH2PO4, 0.5% (NH4)2SO4, 0.6 g/L MgSO4.7H2O, 0.6 g/L CaCl2.2H2O, 5 mg/L FeSO4.7H2O, 1.6 mg/L MnSO4.H2O, 1.4 mg/L ZnSO4.7H2O, and 3.7 mg/L CoCl2.6H2O) was inoculated with the spore suspension, and the Chit33 production was checked every 24 h using western blotting (see later discussion). After 48 h of growth, 4-L Trichoderma minimal medium + 0.2% proteose peptone was inoculated with the preculture and grown for 96 h at 28°C. Glucose levels were monitored during the growth, and glucose was added when necessary. The culture supernatant was harvested by filtering the culture over a Whatman GF/B filter and frozen at –20°C.

Purification of the Chit33 and Chit42
The pH of the culture supernatant from the 42-kDa chitinase (1200 ml) was adjusted by adding 80 ml 1 M KPi, pH 7.5, and loaded onto a DEAE Sepharose FF column (Pharmacia, Uppsala, Sweden; 2.5 x 15 cm), which was equilibrated with 25 mM KPi, pH 7.5, at a flow rate of 1 ml/min. After washing the column until the A280 of the effluent reached the value of the equilibration buffer, the protein was eluted with a linear gradient (2 x 300 ml) of 0–1 M NaCl. Fractions were collected and Chit42 was detected by measuring the activity on MeUmb-NAG2 (see later description). The Chit42-containing fractions were pooled and concentrated, and the buffer exchanged by ultrafiltration (PES membrane with 10-kDa cut-off, Vivascience, Hanover, Germany) to 25 mM KPi, pH 6.5. The concentrated sample was further purified using a P-60 gel filtration column (BioRad, Richmond, CA; 1 x 30 cm) at a flow rate of 4 ml/h. After gel filtration, the Chit42-containing fractions were pooled and concentrated by ultrafiltration.

The purification of the Chit33 was started by concentrating the 4-L culture supernatant to 400 ml and at the same time changing the buffer to 25 mM citrate buffer, pH 3, by ultrafiltration (PES membrane with 10 kDa cut-off, Vivascience). The concentrated supernatant was loaded onto a SP-Sepharose FF column (Pharmacia; 2.5 x 10 cm) equilibrated with 25 mM citrate buffer, pH 3.0, and the column was washed with the equilibration buffer at a flow rate of 1 ml/min until the baseline was reached. The column was eluted with a linear gradient (2 x 200 ml) of 0–0.5 M NaCl. Fractions were collected and screened for the presence of Chit33 by western blot analysis (see later description). The Chit33-containing fractions were pooled and concentrated, and the buffer exchanged by ultrafiltration to 25 mM KPi, pH 6.5, and the sample loaded onto a Biogel P-60 gel filtration column (BioRad; 1 x 30 cm) at a flow rate of 4 ml/h. After gel filtration the Chit33-containing fractions were pooled and concentrated by ultrafiltration.

Protein analysis
Protein concentrations after purification were determined by the BCA method (Pierce, Rockford, IL) using bovine serum albumin as the standard (Smith et al., 1985Go). The purity of the enzymes was checked on SDS–PAGE gels stained with Coomassie brilliant blue (Laemmli, 1970Go). For western blot analysis rabbit polyclonal antibodies were used to detect Chit33 or Chit42 (Towbin et al., 1979Go). The N-terminal amino sequences of the purified protein preparations were determined by Edman degradation using an Applied Biosystems (Foster City, CA) 949A Procise protein sequencer (Hewick et al., 1981Go).

Kinetic and inhibition studies using chromophoric and fluorogenic chito-oligosaccharides
Soluble NAGn-MU (Sigma, St. Louis, MO) and NAGn-PNP (Seikagaku, Tokyo) were used for kinetic characterisation of the two enzymes. The initial rates in the presence or absence of inhibitors were determined by measuring product formation at different time points after the reaction was stopped with 0.5 M Na2CO3. All assays were performed at 30°C. The inhibition of Chit33 and Chit42 was tested with the following compounds: allosamidin, O-(chitobiosylidene) amino N-phenylcarbamate, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate, NAG2, NAG3 and NAG4. The more detailed assay conditions used in the various hydrolysis experiments are stated in the text or figure legends. For NAGn-MU cleavage, the increase in the fluorescence (i.e., the release of the MU) was measured using a Wallac Victor2 V (Turku, Finland) microtiter plate reader (excitation wavelength at 355 nm and emission wavelength at 460 nm). For NAGn-PNP cleavage, the release of the para-nitrophenol group was measured at 405 nm with a microtiter plate reader. Steady-state kinetic parameters are derived from fitting the data to the Michaelis-Menten equation using Microcal Origin 6.0 software (Northampton, MA). The enzyme concentration used in the NAGn-PNP assays was 0.5–1.5 µM and in the more sensitive fluorescent NAGn-MU assays 10–50 nM. The detailed degradation patterns of different NAGn-PNPs were analyzed by HPLC equipped with a UV detector. The enzymatic reactions were stopped with 0.5% H3PO4 and analyzed with a Ultrahydrogel 120 column (Waters, Milford, CT) using isocratic elution with 0.1% H3PO4. GlcNAc mono- and oligomers were detected at 210 nm and free PNP groups at 300 nm.

Chemoenzymatic synthesis of ß-1,4-galactosylated and {alpha}-1,3-fucosylated chito-oligosaccharides
Enzymatic synthesis of ß-1,4-galactosylated chito-oligosaccharides (Galß1-4GlcNAcß1-4R) was performed as described by Brew et al. (1968)Go. Enzymatic synthesis of {alpha}-1,3-fucosylated chito-oligosaccharides (GlcNAcß1-4(Fuc{alpha}1-3)GlcNAcß1-4GlcNAcß1-4R) was performed as described by Natunen et al. (2001)Go. Fuc-NAG6 and Fuc-NAG4 were produced by {alpha}3fucosyltransferase reaction (human recombinant Fuc-TV, Calbiochem, Darmstadt, Germany), purified by gel filtration (Superdex Peptide HR 10/30, Pharmacia) and high-pH anion exchange chromatography (CarboPac PA-1 column, Dionex, Sunnyvale, CA) separating nonreacted acceptor and characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and 1D and 2D NMR as described in Natunen et al. (1999Go, 2001)Go. The fucosylated oligosaccharides were at least 95% pure, Fuc-NAG6 product containing about 96% of the heptasaccharide and about 4% of NAG6 (Natunen et al., 1999Go). Gal-NAG6 and Gal-NAG4 were produced in quantitative ß4galactosyltransferase reaction and purified by gel filtration (Superdex peptide 10/30, Pharmacia) and analyzed by MALDI-TOF MS showing single products without oligosaccharide impurities (Natunen et al., 1999Go).

MS analysis and quantification of the hydrolysis products
MALDI-TOF MS was performed essentially as described in Räbinä et al. (1997)Go, using the positive ion mode and 2,5-dihydroxybenzoic acid as the matrix in a BIFLEX instrument (Bruker-Franzen Analytik, Bremen, Germany). Before MS analysis, at the end of each hydrolysis reaction the oligosaccharides were purified, then the protein was denatured and together with salts removed by two-layer ion exchange chromatography using Dowex AG1-X8 (Ac, 200–400 mesh, BioRad) and Dowex AG50W-X8-(H+, 200–400 mesh, Biorad) (Natunen et al., 2001Go; Räbinä et al., 1997Go).

Binary model mixtures of chitotriose (NAG3) and chitotetraose (NAG4) were prepared from stock solutions containing 5.0 mg pure commercial glycans. Five mixtures containing 10, 25, 50, 75, and 90 mol% of NAG3 were analyzed by MALDI-TOF MS. Quite satisfactory peak height ratios (12, 24, 50, 71, and 92% for NAG3) were observed in all spectra obtained from the model mixtures. The quantities of the oligosaccharide products from the hydrolysis experiments were determined as molar percentages from the heights of the MS peaks. Our data show that the NAG3 and NAG4 peaks are at least semi-quantitatively if not quantitatively comparable to the actual amounts. It has been already shown that oligosaccharides with molecular weight of about 1000 or more give quantitative peaks in MALDI-TOF MS (Naven and Harvey, 1996Go; Nyman et al., 1998Go). This indicates that the data for oligosaccharides of five or more units long (e.g., NAG5, NAG6, Gal-NAG4, etc.) are quantitative. The biggest inaccuracy in quantification comes from the analysis of the NAG2 and especially NAG peaks, because this region contains matrix peaks that may quench the signals, and the peaks may be also overlapped by large matrix signals. The quenching of signal may be observable in Figure 2. There should be equal amount of NAG2 and NAG4 oligosaccharides, but the amount of tetra-saccharides is about 60% whereas there are only 25% of disaccharides. The disaccharide signal is either quenched or the rest of the smaller saccharides are in the form of NAG monosaccharide. The presence of NAG2 signals thus indicates qualitative presence of the disaccharides and likely presence of substantial amount of the disaccharides.

Chitinase model building
Structural models of the T. harzianum Chit33 and Chit42 were built based on the known structures of homologous fungal and plant chitinases from family 18. Sequence comparisons and structural alignments used for model building were made using the Multalin program (Corpet, 1988Go) and ClustalW (Thompson et al., 1994Go). The structure of chitinase-1 (CiX1) from the pathogenic fungus C. immitis (PDB-code 1D2K, Hollis et al., 2000Go) sharing 53% sequence identity with Chit42 and hevamine from Hevea brasiliensis (PDB code 1LLO, Terwisscha van Scheltinga et al., 1995Go) sharing 31% sequence identity with Chit33, served as template structures for model building. Chit33 and Chit42 homology models were constructed using the Insight II Homology program (Accelrys), and they were used as starting point for the construction of models in complex with hexa-N-acetyl-chitohexaose and -heptaose. The quality of the models generated was evaluated with PROCHECK (Laskowski et al., 1993Go). The complex structures were created by superimposing the Chit42 model with the crystal structure of chitinase A from S. marcescens complexed with an octa-acetylchito-octaose (PDB code 1EHN, Papanikolau et al., 2001Go) and the Chit33 model with the hevamine complex structures (PDB codes 1LLO and 1HVQ, Terwisscha van Scheltinga et al., 1994Go, 1995Go) containing allosamidin and tri-N-acetyl-chitotriose. This positioned the chito-oligosaccharides in the active sites of the T. harzianum enzymes, and further energy optimization was performed to refine the complex structures.


    Acknowledgements
 
Dr. Tahía Benítez and Dr. Carmen Limón (University of Sevilla) are thanked for supplying the Chit33 gene of T. harzianum and the polyclonal antibodies against Chit42 and Chit33. We are grateful to Dr. Andrea Vasella (ETH Zürich) for supplying the N-phenylcarbamate derivatives and to Dr. Shohei Sakuda for a sample of allosamidin. Arja Kiema and Kirsi Rautjoki (VTT Biotechnology) are thanked for excellent technical assistance and Tapani Suortti (VTT Biotechnology) for the analysis of oligosaccharides by HPLC. We thank Minna Ekström, Jari Helin. and Ritva Niemelä (University of Helsinki) for the MS analysis of the oligosaccharides. Nisse Kalkkinen (University of Helsinki) is thanked for the N-terminal sequence determination. This research was supported by TEKES, the National Technology Agency of Finland, and it is also a part of the research program VTT Industrial Biotechnology (Academy of Finland; Finnish Centre of Excellence program, 2000–2005, project no. 64330).


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: harry.boer{at}vtt.fi

2 Present address: Glykos Finland ltd, Viikinkaari 4, 00790 Helsinki, Finland Back

3 Present address: Orion Pharma, P.O. Box 65, FIN-02101 Espoo, Finland Back


    Abbreviations
 
HPLC, high-performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectroscopy; MU, 4-methylumbelliferyl; NAG, N-acetyl-ß-D-glucosamine; NMR, nuclear magnetic resonance; PNP, p-nitrophenyl; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


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 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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