Eastberg Jennifer H, Eklund Jennifer, Monnat Raymond, Stoddard Barry L
Fred Hutchinson Cancer Research Center and Graduate Program in Molecular and Cellular Biology, University of Washington, 1100 Fairview Avenue North, A3-025, Seattle, Washington 98109, USA.
Biochemistry. 2007 Jun 19;46(24):7215-25. doi: 10.1021/bi700418d. Epub 2007 May 22.
Several unique protein folds that catalyze the hydrolysis of phosphodiester bonds have arisen independently in nature, including the PD(D/E)XK superfamily (typified by type II restriction endonucleases and many recombination and repair enzymes) and the HNH superfamily (found in an equally wide array of enzymes, including bacterial colicins and homing endonucleases). Whereas the identity and position of catalytic residues within the PD(D/E)XK superfamily are highly variable, the active sites of HNH nucleases are much more strongly conserved. In this study, the ability of an HNH nuclease to tolerate a mutation of its most conserved catalytic residue (its histidine general base), and the mechanism of the most active enzyme variant, were characterized. Conversion of this residue into several altered chemistries, glutamine, lysine, or glutamate, resulted in measurable activity. The histidine to glutamine mutant displays the highest residual activity and a pH profile similar to that of the wild-type enzyme. This activity is dependent on the presence of a neighboring imidazole ring, which has taken over as a less efficient general base for the reaction. This result implies that mutational pathways to alternative HNH-derived catalytic sites do exist but are not as extensively or successfully diverged or reoptimized in nature as variants of the PD(D/E)XK nuclease superfamily. This is possibly due to multiple steric constraints placed on the compact HNH motif, which is simultaneously involved in protein folding, DNA binding, and catalysis, as well as the use of a planar, aromatic imidazole group as a general base.
自然界中已经独立出现了几种催化磷酸二酯键水解的独特蛋白质折叠结构，包括PD(D/E)XK超家族（以II型限制性核酸内切酶以及许多重组和修复酶为代表）和HNH超家族（存在于同样广泛的一系列酶中，包括细菌的大肠杆菌素和归巢核酸内切酶）。虽然PD(D/E)XK超家族中催化残基的身份和位置高度可变，但HNH核酸酶的活性位点保守性要强得多。在本研究中，对一种HNH核酸酶耐受其最保守催化残基（其组氨酸通用碱）突变的能力以及最具活性的酶变体的机制进行了表征。将该残基转化为几种化学性质改变的氨基酸，谷氨酰胺、赖氨酸或谷氨酸，会产生可测量的活性。组氨酸到谷氨酰胺的突变体表现出最高的残余活性，并且其pH谱与野生型酶相似。这种活性依赖于相邻咪唑环的存在，该咪唑环已作为反应效率较低的通用碱。这一结果表明，通向替代HNH衍生催化位点的突变途径确实存在，但在自然界中，与PD(D/E)XK核酸酶超家族的变体相比，其分化或重新优化的程度没有那么广泛或成功。这可能是由于对紧凑的HNH基序施加了多种空间限制，该基序同时参与蛋白质折叠、DNA结合和催化，以及使用平面芳香咪唑基团作为通用碱。
