Zhang S, Irwin D C, Wilson D B
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Eur J Biochem. 2000 Jun;267(11):3101-15. doi: 10.1046/j.1432-1327.2000.01315.x.
Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.
构建、克隆了栖热放线菌Cel6B六个环残基中的15个突变基因以及五个保守非催化活性位点残基中的8个突变基因，并在大肠杆菌或变铅青链霉菌中进行表达。对突变酶进行了羧甲基纤维素（CMC）、膨胀纤维素（SC）、滤纸（FP）和细菌微晶纤维素（BMCC）以及纤维四糖、纤维五糖和2,4 -二硝基苯基 -β -D -纤维二糖的催化活性测定。还对它们进行了配体结合、酶持续合成能力、热稳定性和纤维二糖反馈抑制的测定。发现两个在两个形成通道的环之间形成二硫键的双Cys突变显著削弱了与配体的结合，降低了所有活性和持续合成能力，表明这些环的运动对Cel6B功能很重要，但不是必需的。两个单突变酶G234S和G284P对SC和FP具有更高的活性，双突变酶对这些底物的活性分别高出三倍和两倍。然而，这些突变酶与内切纤维素酶栖热放线菌Cel5A的协同作用并未增加。所有对滤纸、BMCC和SC活性较低的突变酶持续合成能力也较低。对于CMC并非如此，这表明Cel6B中的持续合成能力是不溶性和结晶纤维素水解的关键因素。位于假定糖基底物亚位点 -2、+1和 +2附近的三个突变（E495D、H326A和W329C）被发现显著增加了对纤维二糖反馈抑制的抗性。A229V和L230C突变均特异性降低了对BMCC的活性，表明BMCC水解具有与其他底物不同的限速步骤。大多数突变酶的热稳定性降低，尽管Cel6B G234S保持了野生型热稳定性。不同突变酶的特性为深入了解Cel6B的催化机制提供了线索。
