Tài liệu Báo cáo khoa học: The role in the substrate specificity and catalysis of residues forming

The role in the substrate specificity and catalysis of

residues forming the substrate aglycone-binding site

of a b-glycosidase

Lu´ cio M. F. Mendonc¸a and Sandro R. Marana

Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil

The b-glycosidases from family 1 of the glycoside

hydrolases are widely distributed among living organ￾isms, being found in bacteria, archea and eukaria.

These enzymes are involved in a high diversity of physi￾ological roles [1,2]. b-glycosidases catalyze the hydro￾lytic removal of the monosaccharide from the

non-reducing end of b-glycosides [2,3]. Their active site

may be divided into subsites, which are of sufficient size

to bind a monosaccharide unit. The monosaccharide

forming the non-reducing end of the substrate, called

glycone, is bound at subsite )1, whereas the remaining

part of the substrate, called aglycone, interacts with the

aglycone-binding site, which may be composed of

several subsites identified by positive numerals. The

substrate is cleaved between subsites )1 and +1 [4].

b-glycosidases are active upon a broad range of sub￾strates, as evidenced by a total of 15 different EC

numbers grouped in family 1 of the glycoside hydro￾lases. Fucose, glucose, galactose, mannose, xylose,

6-phospho-glucose and 6-phospho-galactose are recog￾nized by the b-glycosidase subsite )1. Nevertheless, the

diversity of aglycones is higher, including monosaccha￾rides, oligosaccharides and aryl and alkyl moieties [2].

Furthermore, the aglycone specificity is an important

factor for determining the physiological functions of

b-glycosidases.

Keywords

aglycone; catalysis; glycoside hydrolase;

specificity; b-glycosidase

Correspondence

S. R. Marana, Departamento de Bioquı´mica,

Instituto de Quı´mica, Universidade de Sa˜o

Paulo, CP 26077, Sa˜o Paulo, 05513-970 SP,

Brazil

Fax: +55 11 3815 5579

Tel: +55 11 3091 3810

E-mail: [email protected]

(Received 1 February 2008, revised 4 March

2008, accepted 13 March 2008)

doi:10.1111/j.1742-4658.2008.06402.x

The relative contributions to the specificity and catalysis of aglycone, of

residues E190, E194, K201 and M453 that form the aglycone-binding site

of a b-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investi￾gated through site-directed mutagenesis and enzyme kinetic experiments.

The results showed that E190 favors the binding of the initial portion of

alkyl-type aglycones (up to the sixth methylene group) and also the first

glucose unit of oligosaccharidic aglycones, whereas a balance between

interactions with E194 and K201 determines the preference for glucose

units versus alkyl moieties. E194 favors the binding of alkyl moieties,

whereas K201 is more relevant for the binding of glucose units, in spite of

its favorable interaction with alkyl moieties. The three residues E190, E194

and K201 reduce the affinity for phenyl moieties. In addition, M453 favors

the binding of the second glucose unit of oligosaccharidic aglycones and

also of the initial portion of alkyl-type aglycones. None of the residues

investigated interacted with the terminal portion of alkyl-type aglycones. It

was also demonstrated that E190, E194, K201 and M453 similarly contrib￾ute to stabilize ES

. Their interactions with aglycone are individually

weaker than those formed by residues interacting with glycone, but their

joint catalytic effects are similar. Finally, these interactions with aglycone

do not influence glycone binding.

Abbreviations

BglB, b-glycosidase from Paenibacillus polymyxa; SbDhr1, b-glycosidase from Sorghum bicolor; Sfbgly, b-glycosidase from

Spodoptera frugiperda; ZmGlu1, b-glycosidase from Zea mays.

2536 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS