Sawaya Rana, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Biochemistry. 2003 Jul 15;42(27):8240-9. doi: 10.1021/bi034396d.
RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.
RNA鸟苷酸转移酶是一种关键酶，它催化真核生物mRNA 5'-帽结构合成三个步骤中的第二步。在此，我们对小鼠加帽酶Mce1的鸟苷酸转移酶结构域进行了突变分析。我们在22个氨基酸位点引入了50种不同的突变，并评估了它们对酵母体内Mce1功能的影响。我们确定了16个氨基酸对Mce1活性至关重要（Arg299、Arg315、Asp343、Glu345、Tyr362、Asp363、Arg380、Asp438、Gly439、Lys458、Lys460、Asp468、Arg530、Asp532、Lys533和Asn537），并通过测试保守性取代的影响阐明了结构-活性关系。Mce1的新突变数据，连同酿酒酵母鸟苷酸转移酶先前的突变研究以及小球藻病毒和白色念珠菌鸟苷酸转移酶的晶体结构，提供了构成并稳定活性位点的官能团的连贯图景。我们的结果扩展并巩固了关于RNA加帽酶、DNA连接酶和RNA连接酶催化具有共同结构基础的假说，这些酶构成了一个由一系列保守基序定义的共价核苷酸转移酶超家族。对基序VI突变对体外Mce1鸟苷酸转移酶活性影响的分析突出了Arg530、Asp532、Lys533和Asn537在GTP结合和核苷酸转移中的重要作用。
