PDBsum entry: 2czn

Hydrolase

PDB-id: 2czn

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

103 a.a. *

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

2czn

Name:

Hydrolase

Title:

Solution structure of the chitin-binding domain of hyperthermophilic chitinase from pyrococcus furiosus

Structure:

Chitinase. Chain: a. Fragment: chitin binding domain. Engineered: yes

Source:

Pyrococcus furiosus. Organism_taxid: 2261. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

UniProt:

Q8U1H5 (Q8U1H5_PYRFU)

Seq:

Struc:

Seq:

Struc:

Seq:

717 a.a.

Struc:

103 a.a.*

Key:	PfamA domain	PfamB domain
Secondary structure	CATH domain

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Resolution:

not givenÅ

NMR structure:

38 models

Authors:

T.Uegaki,T.Ikegami,T.Nakamura,Y.Hagihara,S.Mine,T.Inoue, H.Matsumura,M.Ataka,K.Ishikawa

Key ref:

T.Nakamura et al. (2008). Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus.. J Mol Biol, 381, 670-680. [PubMed id: 18582475] [DOI: 10.1016/j.jmb.2008.06.006]

Date:

13-Jul-05

Release date:

18-Jul-06

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1016/j.jmb.2008.06.006

J Mol Biol 381:670-680 (2008)

PubMed id: 18582475

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus.

T.Nakamura, S.Mine, Y.Hagihara, K.Ishikawa, T.Ikegami, K.Uegaki.

ABSTRACT

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.

Selected figure(s)

Figure 1.
Fig. 1. The NMR and crystal structures of ChBD2. (a) The 38 best NMR structures of ChBD2 with the lowest pseudo-energies. These structures are superimposed on the regular secondary structure regions, shown in orange (strands A, B, D and G) and blue (strands C, E, F and H). The figure was made using Molscript.^12 (b) Crystal structure of ChBD2 around Trp308, which forms the chitin-binding surface. Shown is the 2F[o] − F[c] electron density map around Trp308 (cyan) contoured at 1σ. The figure was made using PyMol.^13 (c) Overlay of NMR (orange) and crystal (sky blue) structures (from Pro262 to Ile358) viewed from the same orientation as (a). The side chains of the three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) are shown in red for the NMR structure and in blue for the crystal structure. The figure was made using Molscript.^12 (d) Topological drawing of ChBD2. Strands A, B, D and G constitute a β-sheet (orange), and strands C, E, F and H constitute the other β-sheet (blue). (e) B-factors of C^α atoms (closed circles) and the deviations of the C^α atoms between NMR and crystal structures (open circles) are plotted versus residue number. Eight β-strands (A–H) are shown by bars.

Figure 6.
Fig. 6. CD spectra of ChBD2. Shown are CD spectra of the wild type and E279T:D281N mutant of ChBD2 measured at 25 °C (blue lines) and 85 °C (red lines).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 381, 670-680) copyright 2008.

Figures were selected by an automated process.