The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT–AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco–AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT–AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

钼辅因子 (Moco) 存在于许多对生物过程至关重要的重要酶的活性位点中。 Moco 中螯合钼的二齿配体是吡喃蝶呤二硫烯（钼蝶呤，MPT）。然而，钼酸盐插入 MPT 的机制以及 Moco 在插入吡喃蝶呤钼酶之前的结构均未知。在这里，我们报告了最后的成熟步骤，其中腺苷酸化 MPT (MPT-AMP) 和钼酸盐是底物。拟南芥Mo 插入酶变体 Cnx1E S269D D274S 的 X 射线晶体学鉴定腺苷酸化 Moco (Moco-AMP) 是该反应序列中意想不到的中间体。 X射线吸收光谱揭示了Cnx1E活性位点中Moco的第一个配位球几何结构。我们利用这一结构信息推导出钼酸盐插入 MPT-AMP 的机制。鉴于它们的结构和序列高度相似，我们建议所有真核钼插入酶都采用这种机制。