Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1

Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1

Biochimica et Biophysica Acta 1748 (2005) 180 – 190 http://www.elsevier.com/locate/bba Overexpression and characterization of a novel chitinase from ...

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Biochimica et Biophysica Acta 1748 (2005) 180 – 190 http://www.elsevier.com/locate/bba

Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1 Ingunn A. Hoella, Sonja S. Klemsdalb, Gustav Vaaje-Kolstada, Svein J. Horna, Vincent G.H. Eijsinka,T a

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432 A˚s, Norway b Plant Protection Center, The Norwegian Crop Research Institute, Høgskoleveien 7, 1432 A˚s, Norway Received 3 November 2004; received in revised form 20 December 2004; accepted 11 January 2005 Available online 26 January 2005

Abstract We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5–5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)n ], 4-methylumbelliferyl (4MU) labelled GlcNAc oligomers and h-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K m, 149+/ 29 AM and k cat, 0.0048+/ 0.0005 s-1), and 2) production of relatively large amounts of trimers and tetramers during degradation of h-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the hanomeric configuration. D 2005 Elsevier B.V. All rights reserved. Keywords: Chitin; Chitinase; Chitooligosaccharides; Ech30; Glycoside hydrolase; Trichoderma

1. Introduction Chitinases hydrolyse chitin, an abundant homopolymer of 1,4-h-linked N-acetyl-d-glucosamine (GlcNAc). Chitin is an important structural component of many organisms, occurring in, for example, fungal cell walls [1], shells of crustaceans [2], and insect cuticles [3]. Therefore, chitin degradation and chitinases are important in a wide variety of biological and biotechnological processes, ranging from the exploitation and environmental clean-up of chitinous wastes [4], to human therapy [5], plant defense systems [6] and biological control [7–11].

T Corresponding author. Tel.: +47 64949472; fax: +47 64947720. E-mail address: [email protected] (V.G.H. Eijsink). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.01.002

Based on the sequences and structures of their catalytic domains, chitinases are usually classified into two families of glycoside hydrolases, family 18 and family 19 [12–14]. Family 18 chitinases share a common (h/a)8-barrel catalytic domain but may show differences with respect to the presence of additional domains, the architecture of the substrate binding-cleft and mode of interaction with the polymeric substrate (exo- or endoactivity) [15–20]. Several family 18 chitinases contain one or more additional domains involved in interactions with the substrate. The active site clefts of family 18 chitinases may be shallow and open, as in the endochitinase hevamine [17,21] or deep and almost closed (btunnelQ shape [22]) as in exochitinases such as ChiA and ChiB from Serratia marcescens [16,18]. Trichoderma atroviride P1 (ATCC 74058), previously known as T. harzianum P1, is a soil borne, filamentous

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fungus known as an effective biocontrol agent of several plant-pathogenic fungi [8]. Trichoderma species produce and secrete a number of hydrolytic enzymes, including several chitinases, which are thought to enable Trichoderma to degrade the chitin-containing cell walls of other fungi [23–25]. Fungal chitinases are also important for survival of the producing organism itself, because they are involved in important morphogenic processes such as spore germination, hyphal elongation and hyphal branching [26–28]. The best-studied chitinolytic systems in Trichoderma species are those of T. harzianum and T. artroviride. Genes encoding a 42 kDa endochitinase [29–31], a 33 kDa endochitinase [32], and a 36 kDa endochitinase [33], all belonging to family 18 of glycoside hydrolases, have been cloned and expressed. Also three genes encoding family 20 N-acetyl-h-d-glucosaminidases (exc1 and exc2 [34]; nag1 [35]) have been cloned and expressed. Recently, the DNA sequence of a gene putatively coding for a small, one-domain family 18 chitinase was submitted to GenBank (ech30; Accession No.AY258147; Fig. 1). The most similar chitinase from a Trichoderma species (encoded by chit33 from Trichoderma harzianum) [32]) shows only 29% sequence identity with Ech30. The ech30 gene

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putatively encodes a 290 residue protein preceded by a 19 residue signal sequence and is thus the smallest Trichoderma chitinase described so far. Sequence comparisons revealed clear similarities with family 18 chitinases. The closest relative for which chitinase activity has been experimentally proven is CHIT30 from the entomopathogenic fungus Metarhizium anisopliae (Genbank accession numberAY545982.1; 65% sequence identity). The closest relative with a known crystal structure is hevamine [17] (Fig. 1). As shown in Fig. 1, the sequence of the mature putative gene product contains residues known to be important for catalysis in family 18 chitinases [36], including the characteristic DXDXE motif containing the catalytic glutamate. The Ech30 protein has not been studied in chitin-induced cultures of T. atroviride P1, but it has been shown that the ech30 gene is expressed during certain growth phases (J. Clarke and S.S. Klemsdal, unpublished results). Although several chitinase genes from Trichoderma species now have been cloned, still not much is known about the enzymological properties of the chitinases they encode, despite the fact that these enzymes are potentially powerful biocatalysts with possible applications in both biocontrol and industrial chitin conversion. Here, we report the overexpression,

Fig. 1. Sequence alignment. The complete amino acid sequences of Ech30 and hevamine were aligned on the basis of a multiple sequence alignment of family 18 chitinases, followed by minor manual adjustments based on inspection of the crystal structure of hevamine. The 19 amino acid long N-terminal signal peptide of Ech30 is not shown. The sequence alignment shows that the Ech30 sequence contains the DXDXE motif characteristic for family 18 chitinases (position 140–145; underlined), which includes the catalytic glutamate. The presence of an Asn at position 202 (marked by an arrow) in Ech30 is indicative for family 18 chitinases with acidic pH-optima for activity (see text). Inspection of the structure of hevamine and a three-dimensional model of Ech30 built using Swiss-model ([51]; model based on the alignment shown here and the 1hvq template), showed that the insertions and deletions all occur in loops on the noncatalytic side of the TIM-barrel, that is, far away from the active site groove.

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refolding, purification and in-depth characterization of the Ech30 chitinase.

2. Materials and methods 2.1. DNA techniques The ech30 gene was amplified from genomic DNA from Trichoderma artroviride P1 in a two step recombinant PCR procedure [37] which led to simultaneous removal of a small intron. Three different 5V-fragments, varying in terms of restriction sites present and the presence of a signal peptide (Table 1), were generated with primer 1A or 1B or 1C and 2. Each of these fragments were recombined with a 409-bp 3V fragment generated with primers 3 and 4 (Table 1; see Genbank Accession No.AY258147 for the DNA sequence and details on the intron). PCR reactions were conducted with Pfu DNA polymerase (Promega, Madison, WI, USA) in a PTC-100 Programmable Thermal Cycler (MJ Research, Waltham, MA, USA). The amplification protocol consisted of an initial denaturation cycle of 2 min at 95 8C, followed by 30 cycles of 30 s at 95 8C, 30 s at 55 8C, and 1 min at 72 8C, followed by a final step of 10 min at 72 8C. Amplified fragments were ligated into vector pCRR4Blunt-TOPORZero Blunt TOPO (Invitrogen, Carlsbad, CA, USA). The gene fragments were excised from the TOPO vectors for insertion in an expression vector, using Xho1 and Nde1 for cloning into Nde1-Xho1 digested pRSET B vector (Invitrogen) or XhoI and BspM1 for cloning into NcoI-XhoI digested pETM10 vector (Gqnter Stier, EMBL Heidelberg, Germany). The pETM10

vector contains a N-terminal six-histidine tag. The final constructs were transformed into E. coli BL21Star (DE) (Invitrogen). DNA sequencing was preformed using a BigDyeR Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer/Applied Biosystems, Foster City, CA, USA) and an ABI PRISMR 3100 Genetic Analyser (Perkin Elmer/ Applied Biosystems). 2.2. Production and purification of recombinant protein After a series of initial experiments (see Results section) plasmid pETM10-ech30, containing the ech30 gene without signal peptide and with N-terminal (His)6-tag, was selected for further studies. E. coli BL21Star (DE) transformants containing the pETM10-ech30 construct were grown at 37 8C in LB-medium with 50 Ag ml 1 kanamycin at 225 rpm, to a cell density of 0.6 at 600 nm. Isopropyl-h-dthiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the cells were further incubated for 4 h at 37 8C followed by harvesting by centrifugation (8000 rpm, 8 min at 4 8C). The cell pellet was resuspended in citrate–phosphate buffer pH 6.0, and the cells were lysed by sonication at 20% amplitude with 305 s pulses (with 5 s delay between pulses) on ice, with a Vibra cell Ultrasonic Processor, converter model CV33, equipped with a 3 mm probe (Sonics, Newtown, CT, USA). The inclusion bodies were harvested by centrifugation (15 000 rpm, 20 min at 4 8C) and solubilized in 8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris–HCl, pH 8.0. Non-solubilizable material was removed by centrifugation (15 000 rpm, 10 min at room temperature). The solubilized protein was purified under denaturing conditions (with 8 M urea present) on a 1060 mm Ni-NTA column (Qiagen, Venlo, The Netherlands)

Table 1 List of oligonucleotide primers used Oligo name:

Oligo sequence:

Restriction Comments: endonuclease cleavage site:

Primer 1A (F) 5VGGCATATGTACTTCCGTAAGGCTGCCGCC3V

Nde1

Primer 1B (F)

5VGGCATATGCAACCCTCCGGTCCTG3V

Nde1

Primer 1C (F)

5VGGTTACCTGCAATTCATGCAACCCTCCGGTCCTGAA3V BspM1

Primer 2 (R)

5VGCCAGATTCATTGTCCAAGTCAAAGCCGTCAAGC3V

Primer 3 (F)

5VCTTTGACTTGGACAATGAATCTGGCAACCCC3V

Primer 4 (R)

5VGGCTCGAGTTACAGACCAGGGGCAATCTT3V

Restriction sites are in bold. F: forward primer, R: reverse primer.

Xho1

Primer used to amplify ech30 fragment containing leader peptid sequence, for cloning into vector pRSET B. Primer used to amplify ech30 fragment not containing leader peptid sequence, for cloning into vector pRSET B. Primer used to amplify ech30 fragment not containing leader peptid sequence, for cloning into vector pETM10. Fusion primer used in a two-step recombinant PCR procedure that led to removal of a small intron; used in combination with primer 1A, 1B or 1C to create 5V gene fragment. Fusion primer used in a two-step recombinant PCR procedure that led to removal of a small intron. Used in combination with primer 4 to create 3V gene fragment. Primer used to amplify the 3V ech30 fragment (with primer 3) or to amplify the complete intron-free gene (with primer 1A or 1B or 1C).

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using a flow rate of 2 ml/min. The column was equilibrated in citrate–phosphate buffer pH 6.0, containing 8 M urea and 20 mM imidazole. After loading the solubilized protein, the column was washed with the starting buffer. The His-tagged protein was then eluted with a citrate–phosphate buffer pH 6.0, containing 8 M urea and 100 mM imidazole, and the purified and solubilized protein was refolded by dialysis against 20 mM Tris–HCl, pH 8.0. This procedure enabled production of milligrams amounts of enzyme (approximately 15–20 mg refolded Ech30 pr. liter culture). Pure ChiB from S. marcescens was obtained using overexpression and purification methods described elsewhere [38]. The gene encoding ChiC1 from S. marcescens (Genbank Accession No. AJ630582, [39]) was cloned in vector pRSETB and overexpressed in E. coli BL21 Star (DE3). Enzyme production was induced with IPTG and a periplasmic extract of induced cells was prepared by an osmotic shock procedure, as described previously for other S. marcescens chitinases [38,40]. This extract was used for one-step purification of ChiC1 by hydrophobic interaction chromatography, using a Phenyl-Superose HR 5/5 column in a FPLC system, which is a procedure that works well for all S. marcescens chitinases [38,40].

Ech30 was added to a final concentration of 225 nM, and samples were taken at 0, 30, 60 and 90 min to record release of 4-MU, as described above. Product formation was linear over time in all cases (indicating that the enzyme was remarkably stable at low pH). All measurements were done in triplicate. The activity towards chitin was estimated using a colloidal chitin substrate, CM-Chitin-RBV (LOEWE Biochemica GmbH, Mqnchen). Reactions were conducted in 37.5 mM sodium acetate buffer (pH 4.6 for Ech30 and pH 5.5 for other chitinases) containing 0.6 mg/ml CM-ChitinRBV. After addition of enzyme to final concentrations of 18, 4 and 1 nM for Ech30, ChiB and ChiC1, respectively, the reaction mixtures were incubated at 37 8C for 10 min. Reactions were stopped by adding 25% (v/v) 0.2 M HCl, and stored on ice for 10 min. After centrifugation, the absorbance at 550 nm was measured, using the sample containing no enzyme as a reference. All measurements were done in duplicate. Activities are expressed in arbitrary units; 1 unit equals a DA 550 of 0.1/min.

2.3. Enzymology with chromogenic substrates

A 100 Al reaction mixture containing 200 AM (GlcNAc)4, (GlcNAc)5, or (GlcNAc)6 (Sigma), 0.1 mg ml 1 BSA and 370 nM purified Ech30 in 50 mM NaAcbuffer, pH 4.5, was incubated at 37 8C during periods varying from 0 min (= just after addition of enzyme) to 24 h. Reactions were stopped by cooling on ice, and the reaction mixtures were stored at 20 8C until analysed by HPLC at room temperature using a Gilson HPLC System (Gilson, Middleton, WI, USA) equipped with a Tosoh TSK-Gel amide-80 column (0.46 ID25 cm) (Tosoh Bioscience, Montgomeryville, PA, USA). Ten microliter of the reaction mixture was injected by a 234 autoinjector (Gilson). The liquid phase consisted of 70% acetonitrile, the flow rate was 0.70 ml/min, and eluted oligosaccharides were monitored by recording absorption at 210 nm. In cases where analysis of the anomeric configuration of the newly formed degradation products was desirable, reactions were performed with higher enzyme concentrations (18.5 AM) and short incubation times (approximately 0.5 min). To stabilize the anomeric ratio as fast as possible and to avoid reaching the anomeric equilibrium, reactions were stopped by freezing on nitrogen and samples were stored at 80 8C. Analyses of the degradation of h-chitin were conducted by incubating 100 Al solutions containing 1 mg ml 1 of hchitin (Squid pen h-chitin, 3 Am in size; Seikagaku, Tokyo, Japan) and 370 nM purified Ech30 in 50 mM NaAc-buffer (pH 4.5). The reaction mixtures were incubated at 37 8C and 230 rpm during periods varying from 0 min (= just after addition of enzyme) to 24 h. Reactions were stopped by freezing in liquid nitrogen and stored at 80 8C until they were analyzed by HPLC as described above.

Enzyme kinetics were determined using the (GlcNAc)3 analogue 4-methylumbelliferyl-h-d-N,N’-diacetylchitobioside (4-MU-(GlcNAc)2; Sigma, St Louis, MO, USA) as a substrate in 50 mM citrate–phosphate buffer, pH 4.6. Standard reaction mixtures contained 37 nM Ech30, 0.1 mg ml 1 bovine serum albumine (BSA) (New England Biolabs, Beverly, MA, USA), and 0–250 AM of the substrate. The reaction mixture (total volume of 100 Al) was incubated at 37 8C, and the reaction was stopped by adding 1.9 ml 0.2 M Na2CO3. The amount of 4-MU released was determined by using a DyNA 200 fluorimeter (Hoefer Pharmacia Biotech, San Francisco, CA, USA). For determination of kinetic properties, samples were taken at 0, 30, 60, 90, and 120 min. The production of 4-MU was linear in all cases, thus permitting straightforward calculation of enzyme velocities. All product formation curves obtained by linear regression had correlation coefficients over 0.99. Kinetic parameters were calculated by direct fitting the data to the Michaelis–Menten equation using Hyper [41]. The activity of Ech30 towards 4-MU-(GlcNAc)3 and 4-MU(GlcNAc) was determined using the same reaction conditions as for 4-MU-(GlcNAc)2. Protein concentrations were determined by using the BioRad Protein Assay (Bio-Rad, Hercules, CA, USA) with BSA as a standard [42]. The optimal pH for Ech30 activity was determined using 4-MU-(GlcNAc) 2 as substrate. Solutions of 4-MU(GlcNAc)2 (150 AM) were prepared using 50 mM citrate– phosphate buffers containing 0.1 mg ml 1 BSA, with pH values ranging from pH 2.5–7.0 (0.5 pH unit intervals).

2.4. Analysis of hydrolysis of GlcNAc oligomers and b-chitin

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3. Results and discussion 3.1. Overexpression and purification of Ech30 The N-terminal 19 amino acids of the predicted 309residue product of the ech30 gene have the typical features of a signal peptide [43] and suggest an extracellular location for the protein in T. atroviride P1. Initial attempts to overexpress the complete ech30 gene in E.coli, using pRSET B, yielded inclusion bodies. Determination of the N-terminal amino acid sequence of protein from the inclusion bodies gave a sequence corresponding to residue 1–10 of the predicted primary product of ech30, indicating that the protein was not processed. The ech30 gene was subsequently cloned without this predicted signal peptide sequence, using two different vectors (pRSET B and pETM10). We were not able to produce soluble protein, despite extensive testing of variation of factors such as the IPTG concentration and induction time, the presence/absence of an N-terminal His6-tag, and the growth temperature. A pETM10-based construct, containing the ech30 gene without its putative signal peptide fused to a His6-tag (see Materials and methods for further details) was used for further studies. Refolding of the protein from inclusion bodies could be achieved by a simple dialysis procedure and the refolding yield was close to 100%. Fig. 2 shows an SDS-PAGE analysis of relevant cell extracts and of the refolded protein. Although refolding of Ech30 was simple and almost complete, we cannot exclude that the recombinant enzyme has another specific activity than the natural

enzyme. It has been shown that refolded hevamine has about 80% of the specific activity of the natural enzyme [44]. It is quite likely that the percentage of Ech30 is equally high, since the enzyme displayed activities towards colloidal CM-Chitin-RBV, which were in the same range as the activities of other chitinases studied in our laboratory (27.3, 216 and 15.3 U/nmol for ChiB and ChiC1 from S. marcescens and Ech30, respectively). The more active of the two Serratia enzymes (ChiC1) is an endochitinase, which, in contrast to Ech30, contains two putative chitinbinding domains [39], which are likely to contribute to its high activity [15]. 3.2. pH-activity profile Fig. 3 shows that Ech30 is active in a narrow pH range and has a pH-optimum of 4.5–5.0. Other Trichoderma chitinases have similar pH optima, for example Chit42 from T. harzianum strain P1 (pH 4.0) [45]. From a physiological point of view, these low pH-optima for activity are not unexpected, since Trichoderma species are known to thrive at slightly acidic pH. Interestingly, Ech30 and other chitinases with acidic pHoptima such as hevamine [21] contain an Asn at the position corresponding to position 202 in Fig. 1 (Asn 184 in hevamine). It was recently shown that the presence of an Asp at this position is responsible for the broadness of the pH-activity profile and the activity at neutral pH observed for other family 18 chitinases. Mutation of this Asp into Asn in ChiB from S. marcescens (Asp215) yielded an bacidicQ chitinase with very low activity at pH 7, but considerable (wild-type like) activity at pH 4.5 [36]. 3.3. Enzyme kinetics

Fig. 2. SDS-PAGE of Ech30. Lane 1 and 4, Benchmark ladder (Invitrogen); lane 2, lysate of E. coli harbouring pETM10-ech30; lane 3, lysate of E. coli harbouring pETM10-ech30 and induced by IPTG; lane 5, Ech30 after purification and refolding (3 Ag).

Although several chitinases from Trichoderma have been isolated, detailed kinetic studies of these proteins are scarce. In most analyses published so far colloidal chitin or chitin derivatives were used as substrates. Because of their heterologous nature, these substrates cannot be used to determine K m and k cat. With 4-MU-(GlcNAc)2 as a substrate, Ech30 displayed a normal hyperbolic relationship between the rate of catalysis, v, and the substrate concentration, [S]. However, substrate concentrations higher than 300 AM 4-MU-(GlcNAc)2 inhibited Ech30 (results not shown; substrate inhibition of this kind is quite common for family 18 chitinases; see Refs. [38,46]). Kinetic parameters for Ech30 were determined using 4-MU-(GlcNAc)2 concentrations ranging from 50 to 250 AM, yielding a K m of 149+/ 29 AM, and a k cat of 0.0048+/ 0.0004 s 1. The specific activities at 50 and 250 AM substrate concentration were 72 and 180 nmol/Amol min, respectively. Studies with the tetramer analogue 4-MU-(GlcNAc)3 showed that Ech30 converts this compound to 4-MU and (GlcNAc)3, and to 4-MU-GlcNAc and (GlcNAc)2, with a

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Fig. 3. Dependence of Ech30 activity on pH. The substrate was 4-MU-(GlcNAc)2.

ratio of approximately 1:3. At 50 AM 4-MU-(GlcNAc)3, the specific activity (in terms of 4-MU release only) was in the order of 40 nmol/Amol min. The (GlcNAc)2 analogue 4MU-GlcNAc was very slowly hydrolyzed to 4-MU and GlcNAc, with a specific activity (at 50 AM substrate concentration) in the order of 1 nmol/Amol min. The results with the 4-MU substrates show that Ech30 has a much lower activity towards short substrates than some well-known exochitinases. For example, kinetic studies of the exochitinase ChiB from S. marcescens with 4-MU-(GlcNAc)2 yielded a K m of 34.1+/ 1.4 AM and k cat of 19.1+/ 0.7 s 1 [38]. The exochitinase ChiA from the same bacterium showed sigmoid kinetics, with relevant kinetic parameters being estimated as a [S]0.5=135 AM and k cat=104 s 1 [38]. It is not uncommon for endochitinases to have low or no activity towards short oligosaccharide substrates. For example the well-known endochitinase hevamine (Fig. 1) only cleaves chito-oligosaccharides that are longer than four residues. Even towards pentamer and hexamer substrates, hevamine was found to act quite slowly, the reported kinetic parameters being K m=13.8+/ 0.7 AM and k cat=0.355+/ 0.010 s 1 for (GlcNAc)5, and K m=3.2+/ 0.8 AM and k cat=1.0+/ 0.06 s 1 for (GlcNAc)6 [47]. 3.4. Hydrolysis of natural substrates To characterize the activity of Ech30 on natural substrates and to obtain information on the subsite structure of the active site, we analysed the degradation of GlcNAc oligomers and h-chitin. The results obtained with GlcNAc oligomers are displayed in Figs. 4–6 (these figures are derived from reactions containing identical amounts of enzyme and substrate). Fig. 4 shows that (GlcNAc)6 was hydrolyzed to (GlcNAc)3 + (GlcNAc)3 and (GlcNAc)4 + (GlcNAc)2, with almost equal efficiencies. The resulting trimers and tetramers were hydrolysed further, although with much lower efficiency (especially the trimer). Hydrol-

ysis of (GlcNAc)5 yielded equal amounts of (GlcNAc)3 and (GlcNAc)2, and (GlcNAc)3 was hydrolyzed further to (GlcNAc)2 + GlcNAc at a very low rate (Fig. 5). Comparison of Figs. 4 and 5 (e.g. the 120 min panels) shows that the pentamer was degraded with lower efficiency than the hexamer. Fig. 6 shows that (GlcNAc)4 was hydrolyzed with even lower efficiency than the pentamer. Hydrolysis of the tetramer produced mainly (GlcNAc)2, but small amounts of (GlcNAc)3 and GlcNAc were also detected. Degradation of h-chitin with Ech30 yielded (GlcNAc)4, (GlcNAc)3, (GlcNAc)2 and GlcNAc, with (GlcNAc)2 as the major product (Fig. 7). Taken together, the results show that Ech30 has properties that are characteristic for endochitinases. The enzyme displays low activity towards short oligomeric substrates and activity increases as the oligosaccharides become longer. On the other hand, the activity towards colloidal CM-Chitin-RBV is similar to that of other chitinases. The presence of trimers and tetramers in the reaction mixtures obtained with h-chitin may also be taken to indicate endoactivity. Under the conditions used in this study, the exochitinases ChiA and ChiB from S. marcescens only produce dimers and minor amounts of monomers, whereas the putative endochitinase ChiC1 also produces trimers and tetramers (B. Synstad, S.J. Horn and V.G.H. Eijsink, unpublished results). 3.5. Subsite analysis At equilibrium, the ratio between the a and h anomeric forms of N-acetylchitooligosaccharides is approximately 60:40, as visible in Figs. 4–6. To get more insight into how Ech30 binds substrate, degradation reactions were done under conditions that prevented equilibrium from being reached. Putative subsites on the enzyme are numbered according to standard nomenclature; cleavage occurs between the sugar units bound in subsites 1 and +1 [48].

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Fig. 4. Time course of (GlcNAc)6 degradation and product formation by Ech30 as analyzed by HPLC on a TSK-Gel amide-80 column. The top panel shows a standard mixture of GlcNAc oligomers. The other panels show how the reaction proceeded (incubation times are indicated). Note that each oligosaccharide yields two peaks, representing the a and h anomers. Note the presence of a very small amount of monomer in the sample taken at 24 h.

Fig. 5. Time course of (GlcNAc)5 degradation and product formation by Ech30. See legend to Fig. 4 for explanation. Note the presence of a very small amount of monomer in the sample taken at 24 h.

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Fig. 6. Time course of (GlcNAc)4 degradation and product formation by Ech30. See legend to Fig. 4 for explanation. Note the presence of a very small amount of monomer and trimer in the sample taken at 24 h.

Fig. 7. Time course of product formation upon h-chitin degradation by Ech30. The top panel represents a standard mixture of GlcNAc oligomers.

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Fig. 8. HPLC analysis of anomeric configurations. The top panel shows a standard mixture of GlcNAc oligomers displaying the usual 60:40 ratio between a and h anomers. Panels A, B and C show the result of Ech30-catalyzed partial hydrolysis of (GlcNAc)6, (GlcNAc)5 and (GlcNAc)4, respectively.

The major products formed upon hydrolysis of the hexamer showed a:h ratios of 61:39, 31:69 and 8:92 for (GlcNAc)4, (GlcNAc)3 and (GlcNAc)2, respectively. Thus, the tetramer shows the equilibrium ratio, whereas the dimer and trimer are dominated by the h anomer to different

extends. The ratios show that the substrate either binds in subsites 2 to +4 (yielding a dimer with h configuration only) or in subsites 3 to +3 (yielding two trimers, one of which has the h configuration only). Thus, Ech30 may have at least seven sugar binding subsites ( 3 to +4) as illustrated

Fig. 9. Schematic representation of Ech30-catalyzed hydrolysis of N-acetylglucosamine oligomers [(GlcNAc)n]. Putative sugar binding sites are labeled ( 3) to (+4), and hydrolysis occurs between site ( 1) and (+1). Panels A, B and C show Ech30-catalyzed hydrolysis of (GlcNAc)6, (GlcNAc)5 and (GlcNAc)4, respectively.

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in Fig. 9A. Alternatively, there may be favoured binding modes for the hexamer, in which one or more sugar residues protrude into the solvent, as has been observed for other chitinases [44,49]. The a:h ratio of the reaction products obtained upon hydrolysis of the pentamer was about 6:94 for (GlcNAc)2, whereas for (GlcNAc)3 the ratio was similar to the equilibrium (Fig. 8B). Thus, the pentamer almost exclusively binds in subsites 2 to +3 (Fig. 9B). The tetramer was converted to two dimers, yielding an a:h ratio of 20:80. This ratio indicates that one of the dimers mainly is the h anomer (new reducing end), whereas the other has an a:h ratio close to the equilibrium ratio (Figs. 8C and 9C). Interestingly, Fig. 8C shows that the a:h ratio for the tetramer had changed from about 60:40 (equilibrium ratio) to approximately 78:22 after 0.5 min of the reaction time. This indicates that Ech30 prefers the h-anomeric configuration of (GlcNAc)4 in the +2 subsite (Fig. 9C). Hydrolysis products from the degradation of h-chitin all had the h-anomeric configuration (results not shown, but the trend is visible in, e.g. the 30 min panel in Fig. 7). The fact that Ech30 produced only h-anomeric products, confirms the classification of the enzyme as a family 18 glycoside hydrolase. 3.6. Concluding remarks So far, several chitinases from T. atroviride P1 have been cloned and overexpressed, but little is known about the structure, function and catalytic mechanism of these enzymes. As far as we know, Ech30 is the smallest chitinase described from T. atroviride P1 so far, and the first chitinase from this organism to be characterized in detail. Whereas several chitinases are known to contribute to the wellknown biocontrol properties of T. atroviride [10,11,33], purified Ech30 did not show anti-fungal activity in preliminary tests with three plant pathogenic fungi. Further studies are needed to verify the biological role of Ech30 in mycoparasitism and/or in Trichoderma development. The results presented here indicate that Ech30 is a small endochitinase. Interestingly, Ech30 cleaves natural oligosaccharides in a rather different manner than the best known small endochitinase so far, hevamine, which degrades pentamers to monomers and tetramers, and hexamers to dimers and tetramers [47] (note that the fact that each oligomer was cut once and only at one position permitted. determination of the kinetic parameters described above). Interestingly, hevamine is thought to have six subsites, running from 4 to +2 [17,50]. In contrast, the results for Ech30 do not suggest a binding site for a 4 sugar, while they do suggest binding sites for sugars bound in postion +3 (Fig. 9B) and maybe even position +4 (Fig. 9A). This difference is remarkable, since the sequence alignment of Fig. 1 shows a high degree of similarity between the two enzymes and since structural

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modelling (see legend to Fig. 1) showed that the insertions and deletions visible in the sequence alignment are all in loops on the non-catalytic side of the TIM-barrel. Thus, the observed differences between hevamine and Ech30 must be due to subtle differences in the substrate binding clefts, which can only be resolved after elucidation of the crystal structure of Ech30.

Acknowledgements This work was funded by a grant from the Norwegian Research Council (no. 140/140497). We thank Gqnter Stier for providing us with vector pETM10, Knut Sletten for protein sequencing, and Bjbrnar Synstad, May Bente Brurberg, Jihong Clarke and Daan van Aalten for helpful discussions.

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