Crystallization and preliminary X-ray crystallographic studies of an exo-beta-D-glucosaminidase from Trichoderma reesei.
Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of approximately 105 kDa (Gls93-F1) and approximately 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 A, and diffracted to 1.8 A resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 A, and diffracted to 2.4 A resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.
Sakamoto
Yasumitsu
Y
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.
Ike
Masakazu
M
Tanaka
Nobutada
N
Suzuki
Yoshiyuki
Y
Ogasawara
Wataru
W
Okada
Hirofumi
H
Nonaka
Takamasa
T
Morikawa
Yasushi
Y
Nakamura
Kazuo T
KT
D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006596:Hexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014242:Trichoderma / Q000201:enzymology*
D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase