Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Biochemistry. 2010 Mar 23;49(11):2464-74. doi: 10.1021/bi100036f.
The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a beta-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within beta-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.
20 kDa 的环生芽胞杆菌 Bcx 是一种研究充分的内切木聚糖酶，具有 β-发夹结构，其 N 端和 C 端处于盐桥接触状态。初步实验验证，Bcx 可以通过 PCR 方法进行环状排列，在环区引入新的末端，同时直接或通过一个或两个甘氨酸连接其天然末端。随后，通过对环状 Bcx 基因进行随机 DNase 切割，生成了一个环状排列突变文库，并在刚果红染色的琼脂上筛选木聚糖酶活性。对 35 个独特的活性环状排列突变体的分析表明，尽管许多新的末端如预期的那样位于外环，但相当数量的末端也位于β-折叠内。此外，一些排列将关键催化残基置于或靠近新末端，对活性的有害影响最小，在一种情况下，活性增加了 4 倍。一个突变体的结构通过 X 射线晶体学确定，而另外三个通过 NMR 光谱学进行了探测。这些研究表明，Bcx 的整体构象在环状排列时变化很小，影响主要限于新末端和连接的旧末端附近的局部流动性增加，以及整体热变性稳定性降低。这个环状排列的木聚糖酶文库为该商业上重要的酶的定向进化提供了一组极好的新起点，同时也为使用非天然类似物替代关键催化残基的内含子介导替换提供了有价值的构建体。这些方法应该可以深入了解酶促糖苷水解的机制。
