Qin Zhen, Yan Qiaojuan, Lei Jian, Yang Shaoqing, Jiang Zhengqiang, Wu Shiwang
College of Food Science and Nutritional Engineering, Research and Innovation Center of Food Nutrition and Human Health (Beijing), China Agricultural University, Beijing 100083, People's Republic of China.
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, People's Republic of China.
Acta Crystallogr D Biol Crystallogr. 2015 Aug;71(Pt 8):1714-24. doi: 10.1107/S1399004715011037. Epub 2015 Jul 31.
β-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) family 17 β-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (β/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear β-1,3-glucan and the remaining glucan is transferred to the end of another β-1,3-glucan acceptor. The first crystal structure of a GH family 17 β-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.
β-1,3-葡聚糖基转移酶（EC 2.4.1.-）在支链葡聚糖的形成以及真菌和酵母的细胞壁组装与重排过程中发挥着重要作用。已分别以1.30 Å、2.30 Å和2.27 Å的分辨率解析了来自米黑根毛霉的一种新型糖苷水解酶（GH）家族17 β-1,3-葡聚糖基转移酶（RmBgt17A）及其活性位点突变体（E189A）与两种底物的复合物的晶体结构。RmBgt17A的整体结构具有典型的（β/α）8 TIM桶状折叠。对RmBgt17A与其他GH家族17成员的结构进行了比较：发现RmBgt17A中不存在位于α6螺旋附近区域以及其他GH家族17成员催化裂隙一部分的保守亚结构域。相反，在RmBgt17A亚位点+2的还原端发现了四个暴露于酶表面的氨基酸残基（Tyr135、Tyr136、Glu158和His172），这阻碍了进入催化裂隙。RmBgt17A的这一独特区域使其催化裂隙比其他已报道的GH家族17酶的催化裂隙短。复合物结构还表明RmBgt17A只能提供亚位点-3至+2。这一结构证据清楚地解释了RmBgt17A的催化模式，即层状二糖从线性β-1,3-葡聚糖的还原端释放，剩余的葡聚糖转移到另一个β-1,3-葡聚糖受体的末端。GH家族17 β-1,3-葡聚糖基转移酶的首个晶体结构可能有助于研究GH家族17蛋白的催化机制，并为进一步的酶工程或抗真菌药物筛选提供基础。
