Zheng Baisong, Yang Wen, Wang Yuguo, Feng Yan, Lou Zhiyong
Tsinghua University, Beijing, People's Republic of China.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Mar 1;65(Pt 3):219-22. doi: 10.1107/S1744309109001316. Epub 2009 Feb 12.
FnCel5A, a thermostable endoglucanase, is a member of glycohydrolase family 5 which catalyzes the hydrolysis of cellulose to glucose in the thermophilic bacterium Fervidobacterium nodosum Rt17-B1. FnCel5A is particularly interesting because of its high thermostability (T(opt) = 353 K, half-life 48 h) and its high specific activity towards carboxymethylcellulose. These properties make FnCel5A an attractive target for protein engineering to improve cellulase activity. In order to resolve the crystal structure of FnCel5A and to gain a better understanding of its biological function, recombinant FnCel5A was expressed, purified and crystallized at 291 K using NaH(2)PO(4)/KH(2)PO(4) as a precipitant. A 2.4 A resolution native data set was collected from a single flash-cooled crystal (100 K) using 20%(v/v) glycerol as a cryoprotectant. These crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.5, b = 81.7, c = 85.2 A, alpha = beta = gamma = 90 degrees . One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.5 A(3) Da(-1). A data set was also collected to 1.7 A resolution from a selenomethionyl derivative; it belonged to space group P2(1)2(1)2(1) with the same unit-cell parameters as the native crystals.
FnCel5A是一种耐热内切葡聚糖酶，属于糖水解酶家族5，可催化嗜热细菌诺氏嗜热栖热菌Rt17 - B1中纤维素水解为葡萄糖。FnCel5A因其高耐热性（最适温度T(opt)=353 K，半衰期48小时）以及对羧甲基纤维素的高比活性而格外引人注目。这些特性使得FnCel5A成为蛋白质工程中改善纤维素酶活性的一个有吸引力的目标。为了解析FnCel5A的晶体结构并更好地理解其生物学功能，使用NaH₂PO₄/KH₂PO₄作为沉淀剂，在291 K下表达、纯化并结晶了重组FnCel5A。使用20%（v/v）甘油作为冷冻保护剂，从单个快速冷却的晶体（100 K）收集了分辨率为2.4 Å的天然数据集。这些晶体属于空间群P2₁2₁2，晶胞参数a = 53.5，b = 81.7，c = 85.2 Å，α = β = γ = 90°。每个不对称单元假定存在一个分子，马修斯系数为2.5 ų Da⁻¹。还从硒代甲硫氨酸衍生物收集了分辨率为1.7 Å的数据集；它属于空间群P2₁2₁2₁，具有与天然晶体相同的晶胞参数。
