Ponyi T, Szabó L, Nagy T, Orosz L, Simpson P J, Williamson M P, Gilbert H J
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
Biochemistry. 2000 Feb 8;39(5):985-91. doi: 10.1021/bi9921642.
Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM10s), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabó, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984].
芳香族氨基酸被认为在碳水化合物结合蛋白中起关键作用，通过与靶配体的糖环形成疏水堆积相互作用。10家族纤维素结合模块（CBM10s）存在于荧光假单胞菌纤维素亚种表达的多种纤维素酶和木聚糖酶中，含有两个酪氨酸和三个色氨酸残基，这些残基高度保守。为了研究这些氨基酸是否在荧光假单胞菌纤维素亚种木聚糖酶A（Pf Xyn10A）的CBM10与纤维素的相互作用中起重要作用，在以离散模块形式表达或与Pf Xyn10A催化结构域（CBM10-CD）融合的CBM10中，将这些残基中的每一个都替换为丙氨酸，并评估CBM10-CD突变蛋白结合多糖的能力。数据表明，与野生型蛋白相比，W22A、W24A和Y8A与纤维素的结合非常弱，而Y12A保留了与葡萄糖聚合物相互作用的能力。当将W7A突变引入CBM10时，该蛋白结构域在大肠杆菌中不积累。相反，CBM10-CD的W7A突变体在大肠杆菌中高效表达，尽管该蛋白与纤维素的结合非常弱。野生型CBM10、W22A和W24A的核磁共振谱非常相似，表明这些突变没有显著影响蛋白折叠。用N-溴代琥珀酰亚胺滴定野生型CBM10、W22A和W24A表明，色氨酸22和色氨酸24位于蛋白表面，而色氨酸7被掩埋。总体而言，这些数据表明色氨酸22、色氨酸24和酪氨酸8在Pf Xyn10A CBM10与纤维素的结合中起直接作用。根据CBM10的三维结构对结果进行了讨论[Raghothama, S., Simpson, P. J., Szabó, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984]。
