Tài liệu Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49

Insights into the reaction mechanism of glycosyl hydrolase family 49

Site-directed mutagenesis and substrate preference of isopullulanase

Hiromi Akeboshi1

, Takashi Tonozuka1

, Takaaki Furukawa1

, Kazuhiro Ichikawa1

, Hiroyoshi Aoki1,2,

Akiko Shimonishi1

, Atsushi Nishikawa1 and Yoshiyuki Sakano1

1

Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan;

2

Fuence Co., Shibuya, Tokyo, Japan

Aspergillus niger isopullulanase (IPU) is the only pullulan￾hydrolase in glycosyl hydrolase (GH) family 49 and does not

hydrolyse dextran at all, while all other GH family 49

enzymes are dextran-hydrolysing enzymes. To investigate

the common catalytic mechanism of GH family 49 enzymes,

nine mutants were prepared to replace residues conserved

among GH family 49 (four Trp, three Asp and two Glu).

Homology modelling of IPU was also carried out based on

the structure of Penicillium minioluteum dextranase, and the

result showed that Asp353, Glu356, Asp372, Asp373 and

Trp402, whose substitutions resulted in the reduction of

activity for both pullulan and panose, were predicted to be

located in the negatively numbered subsites. Three Asp￾mutated enzymes, D353N, D372N and D373N, lost their

activities, indicating that these residues are candidates for the

catalytic residues of IPU. The W402F enzyme significantly

reduced IPU activity, and the Km value was sixfold higher

and the k0 value was 500-fold lower than those for the wild￾type enzyme, suggesting that Trp402 is a residue participa￾ting in subsite )1. Trp31 and Glu273, whose substitutions

caused a decrease in the activity for pullulan but not for

panose, were predicted to be located in the interface between

N-terminal and b-helical domains. The substrate preference

of the negatively numbered subsites of IPU resembles that of

GH family 49 dextranases. These findings suggest that IPU

and the GH family 49 dextranases have a similar catalytic

mechanism in their negatively numbered subsites in spite of

the difference of their substrate specificities.

Keywords: dextranase; GH family 49; isopullulanase; pullu￾lan-hydrolase; site-directed mutagenesis.

Isopullulanase (IPU, EC 3.2.1.57; pullulan 4-glucanohydro￾lase) from Aspergillus niger ATCC9642 hydrolyses pullulan

to produce isopanose (Glc-a-(1fi4)-Glc-a-(1fi6)-Glc) and

also hydrolyses substrates containing the panose (Glc-a-

(1fi6)-Glc-a-(1fi4)-Glc) structure, and cleaves the a-1,4-

glucosidic linkage in the panose motif [1,2]. Enzymes that

hydrolyse specific sites of pullulan can be classified into the

following three types (schematic action patterns of these

enzymes have been illustrated previously [2]). (a) Pullulanase

(EC 3.2.1.41), which hydrolyses a-1,6-glucosidic linkages to

produce maltotriose [3]; (b) Thermoactinomyces vulgaris

R-47 a-amylase (TVA, EC 3.2.1.1) [4] and neopullulanase

(EC 3.2.1.135) [5], which hydrolyse a-1,4-glucosidic linkages

to produce panose; and (c) IPU, which hydrolyses the other

a-1,4-glucosidic linkages to produce isopanose. Except for

IPU, these enzymes are classified into glycosyl hydrolase

(GH) family 13, known as the a-amylase family (reviewed in

[6–8]). In contrast, IPU is the sole enzyme classified into GH

family 49 [2,9,10] among these pullulan-hydrolases, and no

homology between IPU and a-amylase family enzymes

has been found (http://afmb.cnrs-mrs.fr/
cazy/CAZY/

index.html).

Interestingly, IPU does not hydrolyse dextran at all, while

all other GH family 49 enzymes are dextran-hydrolysing

enzymes, such as endo-dextranase (EC 3.2.1.11) [11–14] and

isomaltotrio-dextranase (EC 3.2.1.95) [15]. We have repor￾ted the molecular cloning of IPU, and indicated that seven

highly conserved regions are found among the primary

structures of these dextran-hydrolases and IPU [2]. The

expression systems of IPU have been constructed with

eukaryotic hosts Aspergillus oryzae and Pichia pastoris

[2,16]. Recently, a three-dimensional structure of GH family

49 dextranase (Dex49A), which shows a 38% sequence

identity with IPU, has been reported, and the catalytic

domain folds into a right-handed parallel b-helix [17].

Crystal structures of polygalacturonases and rhamnogalac￾turonases, which belong to GH family 28, have been solved

by many researchers (for example [18–20]). Although the

substrate specificities between GH family 49 and 28 are

completely different, the GH family 28 polygalacturonases

and rhamnogalacturonases consist of the similar b-helical

structures, and GH family 49 and 28 form clan GH-N

Correspondence to Y. Sakano, Department of Applied Biological

Science, Faculty of Agriculture, Tokyo University of Agriculture and

Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509 Japan.

Fax: +81 42 367 5705, E-mail: [email protected]

Abbreviations: IPU, isopullulanase; GH, glycosyl hydrolase; BMM,

buffered minimum methanol medium; YPGY, yeast peptone glycerol

medium.

Enzyme: isopullulanase (EC 3.2.1.57); pullulanase (EC 3.2.1.41); neo￾pullulanase (EC 3.2.1.135); R-47 a-amylase (TVA, EC 3.2.1.1); endo￾dextranase (EC 3.2.1.11); isomaltotrio-dextranase (EC 3.2.1.95);

glucoamylase (anomer-inverting enzyme; EC 3.2.1.3); a-glucosidase

(anomer-retaining enzyme; EC 3.2.1.20).

(Received 23 June 2004, revised 16 August 2004,

accepted 27 September 2004)

Eur. J. Biochem. 271, 4420–4427 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04378.x