Safchia Is a New Class of Defence Chitinase from Saffron (Crocus Sativus L

SafchiA is a New Class of Defence Chitinase from Saffron (Crocus sativus L.)

R. Castillo, L. Gómez-Gómez and J.A. Fernández

Laboratorio de Biotecnología

Instituto de Desarrollo Regional

Universidad de Castilla-La Mancha

Campus Universitario s/n

Albacete E-02071

Spain

Keywords: defence, new chitinase, site-directed mutagenesis

Abstract

Plant chitinases (EC 3.2.1.14) catalyse the hydrolysis of chitin, a β-1, 4-linked homopolymer of GlcNac. Chitinases are widely distributed in the plant kingdom and have diverse roles in plant growth and development, as well as in defence responses. We have isolated a chitinase from saffron (Crocus sativus L.) named as SafchiA. A lot of chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. The deduced SafchiA protein share high similarities with chitinases belong to family 19 of glycosyl-hydrolases. Although, some changes in the enzyme catalytic domain are present. The most relevant difference is that one glutamic acid in the enzyme active site described as essential in catalytic event of family 19 glycosyl-hydrolases is not present in SafchiA. Instead a glutamic acid SafchiA posses a tyrosine residue at the corresponding position (Tyr136). In order to gain detailed insight in the role of this and other residues in C. sativus chitinase activity, we have developed a heterologous expression system for SafchiA and different mutated versions of its catalytic domain in E. coli. Using barley class II chitinase as a paradigm, site directed mutagenesis of different active site residues was performed. The abilities of wild type recombinant SafchiA catalytic domain and different mutated versions to hydrolyse the soluble high-molecular-mass substrate CM-chitin-RBV were investigated. Results showed that it seems like only one glutamic acid residue is necessary for the enzyme activity instead of two described in family 19 chitinases.

Introduction

Chitinases, are widely distributed in the plant kingdom and have diverse roles including defence responses (for review, see Meins et al., 1992; Leubner-Metzger and Meins, 1999; Neuhaus, 1999; Gomez et al., 2002; Leubner-Metzger, 2003).

Chitinases have been classified by sequence similarity into seven classes. (Collinge et al., 1993; Neuhaus et al; 1996). Class I, II, IV and VII chitinases are of plant origin and comprise family 19 of glycosyl-hydrolases (Henrissat and Bairoch, 1993), they have been found only in plants and some Streptomyces strains (Watanabe et al., 1999) and their structures are rich in α- helix (Hart et al., 1995) similar to that of hen lysozyme (HEWL).Chitinases of class III, V and VI comprise glycosyl-hydrolases family 18, they are distributed in a wide range of organisms, including bacteria, fungi, plants, insects, mammals and viruses, and posses a common (α/β)8-barrel domain (Hollis et al., 2000). Fam 19 chitinases operate by acidic catalytic mechanisms that involve two glutamic acid residues participation, one that acts as general acid and the other one as general base, through an oxocarbenium glycosyl-enzyme intermediate formation and inversion of the reaction product anomeric conformation. Glycosyl-hydrolases from Fam 18 are retaining enzymes that perform catalysis by the formation and subsequent breakdown of a covalent intermediate species.

It is well known how different active site residues from different glycosil-hidrolases operate during these mechanisms, as class II endochitinase from H. vulgare (Andersen et al., 1997), hevamine (class III chitinase) from Hevea brasiliensis (Bokma et al., 2002) and HEWL (Vocadlo et al., 2001) active sites, which is a very useful tool to deduce amino acid implications of homologues or related enzymes.

Materials and methods

Cloning the Catalytic Domain of SafchiA

For the expression of different versions of the SafchiA catalytic domain, pGex-4T1 (Amersham Biosciences) expression vector was used. This vector allows the expression as Glutathione S-transferase (GST) fusion proteins. Previously the wild type catalytic domain of SafchiA sequence was cloned into pGEM®-T Easy (Promega). The primers used for the PCR amplification were 5´-GATCCGTCATCAGTTCCTCTCAGTTC-3´ with a BamHI restriction site (in italics) for the 5´ end, and 5´- ttgcttaaatatgttccaagctga-3´ for the 3´ end. After the PCR amplification and ligation in pGEM®-T Easy, the construct was digested with BamHI and NotI and the product was then ligated in pGex-4T1 treated with the same restriction enzymes.

Site-directed Mutagenesis

Table 1 gives an overview of the primer pairs that were used for site-directed mutagenesis. Mutants were made by introduction of mutations in one segment of the sequence using a primer flanking the 3’ extreme of SafchiA catalytic domain (see above) and a 5´ internal primer harbouring the mutation, the other fragment amplification was performed using a 5´ external primer and a 3´ internal one, harbouring both of the internal primers nested sequences. Then two fragments were joined by PCR amplification using 5´and 3´ external primers. After cloning in pGEM®-T Easy, propagation in E. coli JM101 cells and plasmid DNA isolation using High Pure Plasmid Isolation Kit (Roche) was done. The mutants were sequenced to check for random PCR mistakes.

Heterologous Expression of SafchiA Catalytic Domain in E. coli

For the heterologous expression of SafchiA catalytic domain variants, E. coli BL21 was used. The bacteria were grown at 37 ºC in 100 ml Luria-Bertani medium. At an O.D600 of 0.6 expression was induced by addition of isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 1 mM; 3 h after induction, bacteria were harvested by centrifugation (5 min, 4 ºC, and 6000 g). After centrifugation, bacterial pellet was suspended in 10 ml Bug Buster® HT Protein Extraction Buffer (Novagen) supplemented with 10 μg/ml lysozyme. After 5 min at 4 ºC inclusion bodies were obtained by centrifugation (30 min, 15,000 g, 4 ºC). The inclusion bodies were washed twice in 50 mM Tris- HCl pH: 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 1 % Triton X-100 and twice in 50 mM Tris HCl pH: 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 0.5 % Sarkosyl. Finally, a third batch of washes was performed without any detergent followed by centrifugation (20 min, 13,000 g, 4 ºC). Pellet was resuspended and incubated during 1 h at 4 ºC in 50 mM HEPES-NaOH pH: 7.5 and 6 M urea until a final protein concentration of 1mg/ml.

Refolding of Inclusion Bodies

The solubilized protein were 10 fold diluted into cold 50 mM HEPES pH: 7.5, 0.2 M NaCl, 1mM DTT, 1M (3(1-pyridinio)-1-propane sulfonate) (NDSB201), then subjected to dialysis against PBS through Amicon® Ultra-4, 30,000 NMWL (Millipore) until a final volume of 1 ml.

Subsequently, 1 ml of the total refolding protein was loaded on a Glutathione Sepharose® 4B (Amersham Biosciences) affinity column and SafchiA and the catalytic domain anchored to Glutathione S-transferase eluted using 10 mM reduced glutathione.

Enzyme Assay

Chitinase activity measurements were performed using a colorimetric assay with CM-chitin-RBV (Loewe Biochemica GmbH) as substrate. The conditions originally described by Wirth and Wolf (1990) were modified to obtain substrate-saturated enzyme reactions and linearity respect the incubation time and the enzyme concentration with the substrate batch and proteins used in the present study. For the assay, protein was diluted with 0.05 M sodium acetate buffer pH: 5.5 to yield a volume of 300 μl. The reaction was started by the addition of 100 µl of 2 mg/ml CM-chitin-RBV, resulting in a final incubation volume of 400 µl. The amounts of proteins used were 0.1μg/ml. After 30 min of incubation at 37 ºC, the reactions were stopped by the addition of 100 µl of 0.2 M HCl and non-degraded material was precipitated on ice overnight. The samples were centrifuged at 10,000 g for 5 min. The absorbance of the supernatant was measured photo-metrically at 550 nm and values were corrected for background (absorbance in the absence of protein). Ten independent measurements were performed and average values used. The enzymatic activity (AE) was defined as A550 /μg/ml protein.

Results

The complete deduced amino acid sequence of SafchiA showed a typical class I chitinase structure (Fig. 2) but comparison of the amino acid sequence with those of previously published chitinases from monocot and dycot plants indicates that SafchiA contained two deletions in the catalytic domain (Fig. 1), one at position 164 and the second one at 172. One of them partially corresponds with the deletion presents in some class II enzymes but larger, the other one is not described in none of the sequences compared. Focussed comparison of the SafchiA catalytic domain sequence with the same domain from the well-known Hordeum vulgare class II chitinase showed that a tyrosine residue occupies the position 136 in SafchiA that corresponds with the Glu67 in barley chitinase. This glutamic acid is described as essential for the enzymatic activity of Fam 19 chitinases because it is the residue that acts as a general acid in the catalysis event (Andersen et al., 1997, García-Casado et al., 1998; Kezuka et al., 2004). Next to this position in the saffron chitinase appears a glutamic acid (Glu149) suitable to act as the proton donor. The other essential residue for the activity of the barley chitinase is the Glu89 which in SafchiA corresponds with Glu159. Other conserved residues are Lys165 and Asn199, these residues are described by Brameld and Godard (1998) as implicated in avoid oxazolinium intermediate formation thus promoting the oxocarbenium formation by establishment of hydrogen bound with the N-acetyl group of the D-sugar C2´.

Alignment by CLUSTALW of deduced amino acid sequence of SafchiA and other class I, II, IV and VII chitinases let us to construct an unrooted phylogenetic tree by Phylodendron informatics program (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html). Tree (Fig. 3) shows that saffron chitinase is not included in any of the discrete groups formed by the different classes of chitinases, being a novel class of Fam 19 chitinases. The abilities of wild type recombinant SafchiA catalytic domain and different mutated versions to hydrolyse the soluble high-molecular-mass substrate CM-chitin-RBV were investigated. Recombinant proteins with specific mutations were expressed, purified and characterized. Using barley class II chitinase as a paradigm, site directed mutagenesis of Glu149 and Glu159 was performed as well as a glutamic residue in Tyr136 position was restored. Results of these analyses are summarizing in table 2. No enzyme activity was detectable for the E159A mutant and the E149L/E159A double mutant. The single E149L mutant retained approximately 86% activity and the T136E mutant showed a slight more activity than the recombinant native version.

Discussion

It is describe that plant chitinases contains conserved residues of cysteine important for its backbone structure and enzymatic activity. These residues established disulfide bonds forming loops in the tertiary structure of the protein. In SafchiA the presence of the main deletion cause the lacking of two cysteine residues in the catalytic domain which causes a difference in the primary structure between this chitinase and other monocot and dicot class I chitinases. This fact let us to considerate SafchiA as a different class of chitinase. Other different clustered deletions have been found in the acidic class I chitinase from Dioscorea japonica (Araki et al., 1992), authors speculate that the loops formed by disulfide bonds of the conserved cysteine are essential for activity of the chitinase; but the absence of the first and second conserved cysteine residues in the catalytic domain of SafchiA is not essential for its activity. We have deepest investigated the role of the saffron chitinase active site. Taking H. vulgare class II chitinase as a paradigm we can observed that one of the conserved glutamic acid residues implicated in the catalytic event of class I and II chitinases are substituted by a tyrosine residue (Tyr136) in SafchiA. Restoration of a glutamic acid in this site as well as mutation of a closer glutamic acid (Glu149) barely affect to the enzyme activity, otherwise mutation of the second glutamic acid (Glu159) implicated in catalysis totally abolished the enzyme activity. It seems like only one glutamic acid residue is necessary for the enzyme activity and it is first described for a class I chitinase and let us to considerate SafchiA to a novel class of chitinase.

ACKNOWLEDGEMENTS

This work is being partly funded by the Consejerías de Educación y Ciencia and Agricultura of the Junta de Comunidades de Castill-La Mancha.

Literature Cited

Andersen, M.D., Jensen, A., Robertus, J.D., Leah, R. and Skriver, K. 1997. Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare). Biochem. J. 322:815-822.

Araki T, Funatsu J, Kuramoto M, Konno H and Torikata T. 1992. The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase. J. Biol. Chem. 267:19944-19947.

Bokma, E., Rozeboom, H.J., Sibbald, M., Dijkstra, B.W. and Beintema, J.J. 2002. Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis. Eur. J. Biochem. 269:893-901.

Brameld, K.A and Goddard, W.A. 1998. The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. Proc. Natl. Acad. Sci. U.S.A. 14; 95:4276-4281.

Collinge, D.B., Kragh, K.M., Mikkelsen, J.D., Nielsen, K.K., Rasmussen , U. and Vad, K. 1993. Plant chitinases. Plant J. 3:31-40.

García-Casado, G., Collada, C., Allona, I., Casado, R., Pacios, L.F., Aragoncillo, C. and Gómez L. 1998. Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds. Glycobiology 8:1021-8.

Gomez, L., Allona, I., Casado, R. and Aragoncillo, C. 2002. Seed chitinases. Seed Sci. Res. 12:217-230.

Hart, P.J., Pfluger, H.D., Monzingo, A.F., Hollis, T. and Robertus, J.D. 1995. The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution. J. Mol. Biol. 248:402-413.

Henrissat, B. and Bairoch, A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293:781-788.

Hollis, T., Monzingo, A.F., Bortone, K., Ernst, S., Cox, R. and Robertus, J.D. 2000. The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis. Protein Sci. 9:544–551.

Kezuka, Y., Kitazaki, K., Itoh, Y., Watanabe, J., Takaha, O., Watanabe, T., Nishizawa, Y. and Nonaka, T. 2004. Crystallization and preliminary X-ray analysis of plant class I chitinase from rice. Protein Pept. Lett.. 11:401-405.

Leubner-Metzger, G. 2003. Functions and regulation of β-1,3-glucanases during seed germination, dormancy release and after-ripening. Seed Sci. Res. 13:17-34.