French Rachel L, Gupta Nirupama, Copeland Paul R, Simonović Miljan
From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 and.
the Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
J Biol Chem. 2014 Oct 17;289(42):28783-94. doi: 10.1074/jbc.M114.597955. Epub 2014 Sep 4.
Selenocysteine (Sec), the 21(st) amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA(Sec)). In archaea and eukaryotes, O-phosphoseryl-tRNA(Sec):selenocysteinyl-tRNA(Sec) synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA(Sec). We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNA(Sec). Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNA(Sec) in vivo. We show that SepSecS preferentially binds one or two tRNA(Sec) molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA(Sec) molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.
硒代半胱氨酸（Sec）是第21种氨基酸，它由丝氨酸前体通过一系列需要硒代半胱氨酸tRNA（tRNA(Sec)）的反应合成。在古细菌和真核生物中，O-磷酸丝氨酰-tRNA(Sec)：硒代半胱氨酰-tRNA(Sec)合成酶（SepSecS）催化末端合成反应，在此反应中，磷酸丝氨酰中间体在连接到tRNA(Sec)的同时被转化为硒代半胱氨酰部分。我们之前已经表明，只有SepSecS四聚体能够结合并识别tRNA(Sec)独特的折叠结构。由于在晶体形式中四个tRNA结合位点中只有两个被占据，因此有人提出疑问，即观察到的排列和结构是否忠实地再现了对硒蛋白形成至关重要的生理相关核糖核蛋白复合物。在此，我们通过使用小角X射线散射、多角度光散射和分析超速离心法确定了人硒代半胱氨酸末端合成复合物的化学计量。此外，我们首次估计了体内SepSecS与tRNA(Sec)之间的比例。我们表明，SepSecS一次优先结合一或两个tRNA(Sec)分子，并且该酶在体内相对于底物tRNA以大摩尔过量存在。此外，我们表明在SepSecS与两个tRNA的复合物中，一个酶同二聚体起到非催化单元的作用，将两个tRNA(Sec)分子的CCA末端定位到另一个催化同二聚体的活性位点凹槽中。最后，我们的结果表明，先前确定的晶体结构代表了生理和催化相关的复合物，并表明SepSecS的变构调节可能在硒代半胱氨酸和硒蛋白合成的调节中起重要作用。
