Llamas Angel, Otte Tanja, Multhaup Gerd, Mendel Ralf R, Schwarz Guenter
Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany.
J Biol Chem. 2006 Jul 7;281(27):18343-50. doi: 10.1074/jbc.M601415200. Epub 2006 Apr 24.
The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn(2+)-/Mg(2+)-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.
钼辅因子（Moco）是通过一条古老且保守的生物合成途径合成的。在植物中，双结构域蛋白Cnx1催化钼插入到钼蝶呤（MPT）中，MPT是一种无金属的磷酸化吡喃蝶呤，带有一个烯二硫醇盐。最近，我们鉴定出一种新型生物合成中间体，腺苷酸化钼蝶呤（MPT-AMP），它是由Cnx1的C端G结构域合成的。在此，我们表明MPT-AMP和钼酸盐以等摩尔且协同的方式结合到另一个N端E结构域（Cnx1E）上。钨酸盐和硫酸盐与钼酸盐竞争，这证明存在一个钼酸盐的阴离子结合位点。Cnx1E催化MPT-AMP的Zn(2+)/Mg(2+)-依赖性水解，但仅当钼酸盐作为共底物结合时才会发生。MPT-AMP水解导致化学计量释放出Moco，其被定量掺入植物脱辅基亚硫酸盐氧化酶中。在形成Moco时，AMP作为反应的第二个产物被释放。当比较MPT-AMP水解与Moco和AMP的形成时，观察到反应速率有1.5倍的差异。结合反应对钼酸盐的严格依赖性，我们提出在核苷酸辅助的向钼蝶呤的金属转移反应中，腺苷酸化钼酸盐作为反应中间体形成。
