Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2012 Nov 2;287(45):38090-100. doi: 10.1074/jbc.M112.404954. Epub 2012 Sep 19.
Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be "tuned" using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein.
翻译后修饰在调节蛋白质结构和功能方面发挥着重要作用。蛋白质法尼基转移酶（FTase）催化多达数百种细胞蛋白质的生物学相关脂化反应。FTase的定点诱变与肽选择性测量相结合表明，分子识别是由多种相互作用共同决定的。对这些相互作用进行靶向随机化产生了具有改变的、在某些情况下具有生物正交选择性的FTase变体。我们证明，可以使用少量在野生型酶中区分非底物时起关键作用的活性位点接触来“调整”FTase的特异性。这种可调节的选择性在体内也能发挥作用，FTase变体能够在细胞内创建具有改变的底物选择性的生物工程平行异戊二烯化途径。工程化的FTase变体为探究异戊二烯化途径酶的选择性以及异戊二烯化途径修饰对蛋白质细胞功能的影响提供了一条新途径。
