Tài liệu Báo cáo khoa học: Three-dimensional structures of thermophilic b-1,4-xylanases from

Three-dimensional structures of thermophilic b-1,4-xylanases

from Chaetomium thermophilum and Nonomuraea flexuosa

Comparison of twelve xylanases in relation to their thermal stability

Nina Hakulinen1

, Ossi Turunen2

, Janne Ja¨ nis1

, Matti Leisola2 and Juha Rouvinen1

1

Department of Chemistry, University of Joensuu, Finland; 2

Helsinki University of Technology, Finland

The crystal structures of thermophilic xylanases from

Chaetomium thermophilum and Nonomuraea flexuosa were

determined at 1.75 and 2.1 A˚ resolution, respectively. Both

enzymes have the overall fold typical to family 11 xylanases

with two highly twisted b-sheets forminga large cleft. The

comparison of 12 crystal structures of family 11 xylanases

from both mesophilic and thermophilic organisms showed

that the structures of different xylanases are very similar. The

sequence identity differences correlated well with the struc￾tural differences. Several minor modifications appeared to be

responsible for the increased thermal stability of family 11

xylanases: (a) higher Thr : Ser ratio (b) increased number of

charged residues, especially Arg, resulting in enhanced polar

interactions, and (c) improved stabilization of secondary

structures involved the higher number of residues in the

b-strands and stabilization of the a-helix region. Some

members of family 11 xylanases have a unique strategy to

improve their stability, such as a higher number of ion pairs

or aromatic residues on protein surface, a more compact

structure, a tighter packing, and insertions at some regions

resultingin enhanced interactions.

Keywords: xylanase; glycoside hydrolases; family 11;

thermostability.

Xylanases (EC 3.2.1.8) are glycoside hydrolases that cata￾lyze the hydrolysis of internal b-1,4 bonds of xylan, the

major hemicellulose component of the plant cell wall. The

enzymatic hydrolysis of xylan has potential economical and

environment-friendly applications. Xylanases can be used in

bleachingof pulp to reduce the use of toxic chlorine￾containingchemicals [1] or to improve the quality of animal

feed [2]. In addition, there are applications in the food and

beverage industry [3]. Therefore, attention is focused on

discovery of new xylanases or improvement of existingones

in order to meet the requirements of industry such as

stability and activity at high temperature and extreme pH.

The xylanases that have been structurally characterized to

date can be classified into the glycoside hydrolase families 10

and 11, correspondingto former families F and G,

respectively [4]. Family 10 enzymes have an (a/b)8 barrel

fold with a molecular mass of approximately 35 kDa.

Family 11 xylanases are somewhat smaller, approximately

20 kDa, and their fold contains an a-helix and two b-sheets

packed against each other, forming a so-called b-sandwich.

Due to the industrial applications of xylanase, both xylanase

families are well studied. In this paper, we focus on

xylanases in family 11.

To date, the crystal structures of family 11 xylanases are

available from several organisms: Trichoderma harzianum

[5], Bacillus circulans [5–7], Trichoderma reesei [8,9], Asper￾gillus niger [10], Thermomyces lanuginosus [11], Aspergillus

kawachii [12], Bacillus agaradhaerens [13], Paecilomyces

varioti [14], and Dictyoglomus thermophilum [15]. Three of

these, T. lanuginosus, P. varioti, and D. thermophilum are

from thermophilic organisms. In addition, a low-resolution

structure has been reported for thermostable Bacillus D3

[16] but no PDB coordinates are available. Very recently,

the structures of two new xylanases from Streptomyces sp.

S38 [17] and Bacillus subtilis B230 [18] have also been solved.

A disulphide bridge has been suggested to be one reason for

the enhanced thermal stability of T. lanuginosus and

P. varioti xylanases [11,14]. A greater proportion of polar

surface and a slightly extended C-terminus together with an

extension of b-strand A5 are thought to increase the stability

of D. thermophilum xylanase [15,19]. Despite all these

studies, the structural basis for the thermostability of family

11 xylanases is not well understood.

We report here the three-dimensional structures of two

new members of family 11 xylanases. The crystal structure

of the catalytic domain from Chaetomium thermophilum

xylanase Xyn11A (CTX) has been determined at 1.75 A˚

resolution and the catalytic domain from Nonomuraea

flexuosa xylanase Xyn11A (NFX) at 2.1 A˚ resolution. CTX

Correspondence to N. Hakulinen, Department of Chemistry,

University of Joensuu, PO Box111, FIN-80101 Joensuu, Finland.

E-mail: [email protected]

Abbreviations: AKX, Aspergillus kawachii xylanase; ANX, Aspergillus

niger xylanase; BAX, Bacillus agaradhaerens xylanase; BCX,

Bacillus circulans xylanase; CTX, Chaetomium thermophilum xylanase;

DTX, Dictyoglomus thermophilum xylanase; GlcNAc, N-acetyl-D￾glucosamine; NFX, Nonomuraea flexuosa xylanase; PVX,

Paecilomyces varioti xylanase; THX, Trichoderma harzianum xylanase;

TLX, Thermomyces lanuginosus xylanase; TRX I, Trichoderma reesei

xylanase I; TRX II, Trichoderma reesei xylanase II.

Enzymes: xylanases (EC 3.2.1.8).

Note: The coordinates of the refined structures have been deposited

with the Protein Data Bank, accession codes are 1H1A for

Chaetomium thermophilum and 1M4W for Nonomuraea flexuosa.

(Received 1 November 2002, revised 17 January 2003,

accepted 3 February 2003)

Eur. J. Biochem. 270, 1399–1412 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03496.x