Baird S D, Hefford M A, Johnson D A, Sung W L, Yaguchi M, Seligy V L
Division of Biological Sciences, National Research Council of Canada.
Biochem Biophys Res Commun. 1990 Jun 29;169(3):1035-9. doi: 10.1016/0006-291x(90)91998-8.
We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.
我们最初以两两比对的方式排列了28种不同的纤维素酶序列，发现其中一半在富含疏水氨基酸的区域含有序列 -Asn-Glu-Pro-。以溶菌酶为模型，谷氨酸残基可能对酶的功能至关重要。我们通过对编码多粘芽孢杆菌和枯草芽孢杆菌内切β-1,4-葡聚糖酶的基因进行定点诱变来测试这种可能性。这两种酶的基因和氨基酸序列显示出很少的相似性。在这些相应的酶中，将Glu-194和Glu-169变为等排谷氨酰胺形式导致羧甲基纤维素酶（CMCase）活性急剧丧失，而通过反向突变可以恢复。对枯草芽孢杆菌酶中保守性较低的残基Glu-72和Glu-147进行类似突变不会导致任何活性丧失。
