Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity
Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity
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DOI：
10.1042/bj3310775
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发表时间：
1998-05-01
影响因子：
4.1
通讯作者：
Gilbert, HJ
中科院分区：
文献类型：
--
作者：
Bolam, DN;Ciruela, A;Gilbert, HJ
为了研究纤维素结合结构域（CBD）的作用方式，荧光假单胞菌（Pseudomonasfluorescenssubsp.）纤维素木聚糖酶A（XYLA（CBD））和纤维素酶E（CELECBD）作为单独的实体表达或融合到热纤梭菌内切葡聚糖酶（EGE）的催化结构域。两种CBD对于细菌微晶纤维素（CELECBD，1.62 X 10（6）M-1; XYLA（CBD），1.83 X 10（6）M-1）和酸溶胀纤维素（CELECBD，1.66 X 10（6）M-1; XYLA（CBD），1.73 X 10（6）M-1）表现出相似的K-a值。用纤维寡糖滴定XYLA（CBD）的NMR谱表明，当CBD与纤维六糖、纤维五糖或纤维四糖结合时，其三个色氨酸残基的环境受到影响。XYLA（CBD）对C-6、C-5和C-4纤维寡糖的K-a值分别为3.3 × 10（2）、1.4 × 10（2）和4.0 × 10（1）M-1，表明CBD可容纳至少6个葡萄糖分子，并且对不溶性纤维素的亲和力比可溶性寡糖高得多。融合的CELECBD或XYLA（CBD）的EGE的催化结构域增强酶的活性对不溶性形式的纤维素，但不对羧甲基纤维素。当CBD与EGE或XYLA的催化结构域一起孵育时，分别以不溶性纤维素和纤维素/半纤维素复合物为底物，没有观察到纤维素酶活性的增加，假单胞菌CBD不诱导分离的植物细胞壁的延伸，也不像一类称为expansins的植物细胞壁蛋白那样削弱纤维素纸条。XYLA（CBD）和CELECBD不从棉花表面释放小颗粒。这些结果与II型CBD的作用模式的意义进行了讨论。
To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA(CBD)) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellum endoglucanase (EGE). The two CBDs exhibited similar K-a values for bacterial microcrystalline cellulose (CELECBD, 1.62 x 10(6) M-1; XYLA(CBD), 1.83 x 10(6) M-1) and acid-swollen cellulose (CELECBD, 1.66 x 10(6) M-1; XYLA(CBD), 1.73 x 10(6) M-1). NMR spectra of XYLA(CBD) titrated with cello-oligosaccharides showed that the envicello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The K-a values of the XYLA(CBD) for C-6, C-5 and C-4 cello-oligosaccharides were estimated to be 3.3 x 10(2), 1.4 x 10(2) and 4.0 x 10(1) M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLA(CBD) to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates, Pseudomonas CBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLA(CBD) and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.