A Structurally Novel Chitinase from the Chitin-Degrading Hyperthermophilic Archaeon Thermococcus chitonophagus

Abstract

A structurally novel chitinase, Tc-ChiD, was identified from the hyperthermophilic archaeon Thermococcus chitonophagus, which can grow on chitin as the sole organic carbon source. The gene encoding Tc-ChiD contains regions corresponding to a signal sequence, two chitin-binding domains, and a putative catalytic domain. This catalytic domain shows no similarity with previously characterized chitinases but resembles an uncharacterized protein found in the mesophilic anaerobic bacterium Clostridium botulinum Two recombinant Tc-ChiD proteins were produced in Escherichia coli, one without the signal sequence [Tc-ChiD(ΔS)] and the other corresponding only to the putative catalytic domain [Tc-ChiD(ΔBD)]. Enzyme assays using N-acetylglucosamine (GlcNAc) oligomers indicated that both proteins hydrolyze GlcNAc oligomers longer than (GlcNAc)4 Chitinase assays using colloidal chitin suggested that Tc-ChiD is an exo-type chitinase that releases (GlcNAc)2 or (GlcNAc)3 Analysis with GlcNAc oligomers modified with p-nitrophenol suggested that Tc-ChiD recognizes the reducing end of chitin chains. While Tc-ChiD(ΔBD) displayed a higher initial velocity than that of Tc-ChiD(ΔS), we found that the presence of the two chitin-binding domains significantly enhanced the thermostability of the catalytic domain. In T. chitonophagus, another chitinase ortholog that is similar to the Thermococcus kodakarensis chitinase ChiA is present and can degrade chitin from the nonreducing ends. Therefore, the presence of multiple chitinases in T. chitonophagus with different modes of cleavage may contribute to its unique ability to efficiently degrade chitin.

Importance: A structurally novel chitinase, Tc-ChiD, was identified from Thermococcus chitonophagus, a hyperthermophilic archaeon. The protein contains a signal peptide for secretion, two chitin-binding domains, and a catalytic domain that shows no similarity with previously characterized chitinases. Tc-ChiD thus represents a new family of chitinases. Tc-ChiD is an exo-type chitinase that recognizes the reducing end of chitin chains and releases (GlcNAc)2 or (GlcNAc)3 As a thermostable chitinase that recognizes the reducing end of chitin chains was not previously known, Tc-ChiD may be useful in a wide range of enzyme-based technologies to degrade and utilize chitin.

FIG 1

Structural features of the ChiD from T. chitonophagus (Tc-ChiD) and truncated Tc-ChiD proteins [Tc-ChiD(ΔS) and Tc-ChiD(ΔBD)]. A putative signal sequence (pink, residues 1 to 25), two chitin-binding domains (ChBDs) (yellow, residues 33 to 133 and 181 to 280), and a putative catalytic domain (green, residues 321 to 805) are indicated.

FIG 2

SDS-polyacrylamide gel electrophoresis of Tc-ChiD(ΔS) and Tc-ChiD(ΔBD) purified from recombinant E. coli strains. Purified Tc-ChiD(ΔS) (87.8 kDa) and Tc-ChiD(ΔBD) (56.1 kDa) were applied (1 μg each).

FIG 3

TLC of hydrolysis products of various GlcNAc oligomers incubated with Tc-ChiD(ΔS) (A) or Tc-ChiD(ΔBD) (B). Reaction mixtures containing 1.4 g liter⁻¹ substrate in 50 mM sodium phosphate buffer (pH 5.0) were incubated with either Tc-ChiD(ΔS) or Tc-ChiD(ΔBD) (6.8 μM each) at 85°C for the indicated time periods (0 to 120 min). Lanes M, standard GlcNAc oligomers ranging from GlcNAc to (GlcNAc)₆ (G1 to G6); lanes b, blank control incubated without enzyme for 120 min at 85°C.

FIG 4

TLC analysis of reaction products of colloidal chitin incubated with Tc-ChiD(ΔS) or Tc-ChiD(ΔBD). Reaction mixtures containing 0.2% (wt/vol) colloidal chitin as a substrate in 50 mM sodium phosphate buffer (pH 7.0) were incubated with either Tc-ChiD(ΔS) or Tc-ChiD(ΔBD) (0.67 μM) at 85°C for the indicated time periods (0 to 36 h). The reaction products were analyzed after centrifugation. Lanes M, standard GlcNAc oligomers ranging from GlcNAc to (GlcNAc)₆ (G1 to G6); lanes b, blank control incubated without enzyme for 36 h at 85°C.

FIG 5

TLC of hydrolysis products of various p-nitrophenyl GlcNAc oligomers incubated with Tc-ChiD(ΔS) (A) or Tc-ChiD(ΔBD) (B). Reaction mixtures containing 1 mM substrate in 50 mM sodium phosphate buffer (pH 5.0) were incubated with either Tc-ChiD(ΔS) or Tc-ChiD(ΔBD) (6.8 μM) at 85°C for the indicated time periods (0 to 120 min). Lanes M, standard GlcNAc oligomers ranging from GlcNAc to (GlcNAc)₆ (G1 to G6); lanes Mp, standard p-nitrophenyl GlcNAc oligomers ranging from pNP-(GlcNAc)₂ to pNP-(GlcNAc)₅ (pNP-G2 to pNP-G5); lanes b, blank control incubated without enzyme for 120 min at 85°C.

FIG 6

(A) Effects of temperature on the activities of Tc-ChiD(ΔS) and Tc-ChiD(ΔBD). The chitinase activities were determined at different temperatures (50 to 95°C) by using the Schales procedure, as described in Materials and Methods. (B) Effects of pH on the activities of Tc-ChiD(ΔS) and Tc-ChiD(ΔBD). The chitinase activities were determined at different pH conditions, as described in Materials and Methods. Symbols: dark green squares, sodium citrate (pH 3.0 to 5.0); blue triangles, sodium phosphate (pH 4.5 to 6.0); light green diamonds, MES (pH 6.0 to 7.0). (C) Thermostabilities of Tc-ChiD(ΔS) and Tc-ChiD(ΔBD). Purified enzymes were incubated in 50 mM Tris-HCl (pH 7.5) supplemented with 150 mM NaCl at 80°C (blue diamonds), 90°C (red squares), and 100°C (green triangles) for various time intervals. The residual activities were determined at 85°C.

FIG 7

Schematic illustration of the reaction catalyzed by Tc-ChiD. Individual GlcNAc monomers are represented by hexagons. The black hexagons indicate the reducing ends of the GlcNAc oligomers, and those in the pNP-GlcNAc oligomers are in gray. Thin arrows indicate positions with lower activities. Asterisks indicate cleavage sites that cannot be distinguished in the experiments.