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, 65 (12), 5338-44

A Unique Chitinase With Dual Active Sites and Triple Substrate Binding Sites From the Hyperthermophilic Archaeon Pyrococcus Kodakaraensis KOD1

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A Unique Chitinase With Dual Active Sites and Triple Substrate Binding Sites From the Hyperthermophilic Archaeon Pyrococcus Kodakaraensis KOD1

T Tanaka et al. Appl Environ Microbiol.

Abstract

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.

Figures

FIG. 1
FIG. 1
Nucleotide and deduced amino acid sequences of the chiA gene. The putative Shine-Dalgarno (SD) sequence is double underlined. The translation termination codon is designated by an asterisk. A putative signal sequence is underlined. CBDs are boxed. Proline- and hydroxyamino acid residue-rich regions are shown by dotted lines.
FIG. 2
FIG. 2
Structural features of the chitinase from P. kodakaraensis (ChiA) and schematic drawings of deletion mutants. A putative signal sequence, regions A and B, three CBDs, and three proline- and hydroxyamino acid residue-rich regions are shown as indicated.
FIG. 3
FIG. 3
(A and B) Two putative active sites of ChiA. Amino acid sequences of two putative active sites of regions A and B are aligned with respective similar amino acid sequences of the active sites of B. circulans (B.cir) chitinase A1 (A) and S. erythraeus (S.ery) chitinase (B). Consensus residues among family 18 bacterial chitinases are indicated above the alignments. Conserved aromatic residues are indicated by “@.” Glutamate residues that probably act as proton donors are indicated by asterisks. (C) Comparison of amino acid sequences among ChiA CBD1, C. paraputrificum (C.par) chitinase B chitin binding domain, and E. chrysanthemi (E.chr) EGZ cellulose binding domain. Identical amino acids are shown against a black background. Two solvent-exposed aromatic residues in the cellulose binding domain of EGZ are indicated by squares and asterisks. Hydrophobic core residues in the cellulose binding domain of EGZ are squared with arrows. (D) Comparison of amino acid sequences among ChiA CBD2, CBD3, and B. fibrisolvens (B.fib) H17c endoglucanase cellulose binding domain. Identical amino acids are shown against a black background. Four strictly conserved tryptophan residues of family II cellulose binding domains are squared with asterisks.
FIG. 4
FIG. 4
SDS-PAGE of purified ChiA and deletion mutants. Lane 1, molecular mass standards, namely, rabbit muscle phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30.1 kDa), and soybean trypsin inhibitor (20.1 kDa); lane 2, purified ChiA (131,235 Da); lane 3, purified ChiAΔ1 (97,553 Da); lane 4, purified ChiAΔ2 (70,567 Da); lane 5, purified ChiAΔ3 (58,871 Da); lane 6, purified ChiAΔ4 (33,832 Da).
FIG. 5
FIG. 5
TLC of restriction products by ChiA from various N-acetyl-chitooligosaccharides. The reaction mixture (50 μl) containing 0.7 mg of substrates in 70 mM sodium acetate buffer (pH 5.0) was incubated with enzyme (GlcNAc1–3, 0.45 μg; GlcNAc4–6, 0.9 μg) at 80°C. The reaction products at 0, 5, 15, 30, 60, and 120 min were analyzed. Lanes C, negative control incubated without enzyme at 80°C for 120 min; lanes S, standard N-acetyl-chitooligosaccharides ranging from GlcNAc (G1) to GlcNAc6 (G6).
FIG. 6
FIG. 6
TLC of restriction products by ChiA from colloidal chitin. The reaction mixture (1 ml) containing 0.16 mg of colloidal chitin in 50 mM sodium acetate buffer (pH 5.0) was incubated with enzyme (0.6 μg) at 80°C for 1.5, 3.0, and 4.5 h. The reaction products (250 μl each) were centrifuged, and the supernatants were concentrated and analyzed. Lane C, negative control incubated without enzyme at 80°C for 4.5 h; lane S, standard N-acetyl-chitooligosaccharides ranging from GlcNAc (G1) to GlcNAc5 (G5).
FIG. 7
FIG. 7
Reaction profiles with various 4-methylumbelliferyl saccharides (GlcNAc1–3-4MU, 350-nm excitation, 440-nm emission). The enzyme reaction mixture containing 10 μl of 1 mM GlcNAc1–3-4MU, 990 μl of 100 mM acetate buffer, and 20 μl of enzyme solution with 18 ng of ChiA was incubated at 80°C. The reaction mixture (100 μl) was added to 900 μl of ice-cold 100 mM glycine-NaOH (pH 11) to terminate the reaction. Liberated 4MU was detected at a wavelength of 440 nm. The rate of 4MU liberation was calculated from the initial reaction velocity. ●, GlcNAc-4MU; ○, GlcNAc2-4MU; ■, GlcNAc3-4MU.
FIG. 8
FIG. 8
Chitin binding assay of ChiAΔ2 and ChiAΔ4. Lanes 1 and 5, enzyme extract used for chitin binding experiment; lanes 2 and 6, unbound fraction to colloidal chitin; lanes 3 and 7, bound fraction to colloidal chitin; lanes 4 and 8, molecular mass standards, namely, rabbit muscle phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), and bovine erythrocyte carbonic anhydrase (30.1 kDa).

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