Henshaw Joanna, Horne-Bitschy Ami, van Bueren Alicia Lammerts, Money Victoria A, Bolam David N, Czjzek Mirjam, Ekborg Nathan A, Weiner Ronald M, Hutcheson Steven W, Davies Gideon J, Boraston Alisdair B, Gilbert Harry J
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
J Biol Chem. 2006 Jun 23;281(25):17099-17107. doi: 10.1074/jbc.M600702200. Epub 2006 Apr 6.
Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.
碳水化合物识别对于植物结构多糖的生物学和工业利用至关重要。这些不溶性聚合物难以被微生物降解，催化这一过程的酶通常包含非催化性碳水化合物结合模块（CBMs），它们通过增加底物结合来增强活性。琼脂糖是二糖3,6-脱水-α-L-半乳糖-（1,3）-β-D-吡喃半乳糖-（1,4）的重复结构，是海藻中的主要基质多糖，但此前尚未探索CBMs在这种重要聚合物水解中的作用。在这里，我们表明存在于两种不同β-琼脂糖酶中的6家族CBMs特异性结合琼脂糖链的非还原端，仅识别二糖的第一个重复结构。其中一个模块Aga16B-CBM6-2与新琼脂六糖形成复合物的晶体结构揭示了该蛋白表现出精确特异性的机制，即靶向脱水-L-半乳糖的赤道O4和轴向O3。将CBM6靶向琼脂糖链的非还原端可能会将附加的催化模块引导至植物细胞壁中被β-琼脂糖酶攻击的区域，在这些区域基质多糖可能更易于进一步的酶促水解。
