Lodeiro Silvia, Schulz-Gasch Tanja, Matsuda Seiichi P T
Department of Chemistry and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
J Am Chem Soc. 2005 Oct 19;127(41):14132-3. doi: 10.1021/ja053791j.
Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Described herein are experiments in which cycloartenol synthase was redesigned to become a highly accurate lanosterol synthase. Several single mutants have been described that modify the catalytic specificity of cycloartenol to form some lanosterol. Modeling studies were undertaken to identify combinations of mutations that cooperate to decrease the formation of products other than lanosterol. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation.
改变酶催化特异性的研究始终表明，相较于限制具有宽泛选择性的酶的选择性，拓宽精确酶的底物或产物特异性更为容易。本文描述了将环阿屯醇合酶重新设计为高度精确的羊毛甾醇合酶的实验。已报道了几个改变环阿屯醇催化特异性以形成一些羊毛甾醇的单突变体。开展了建模研究以确定协同作用减少羊毛甾醇以外产物形成的突变组合。构建并表征了一个双突变体，结果表明其能将氧化角鲨烯精确环化生成羊毛甾醇（99%）。这种催化变化既通过His477Asn突变改变了极性，又通过Ile481Val突变改变了空间限制。
