Santos-Silva Teresa, Ferroni Felix, Thapper Anders, Marangon Jacopo, González Pablo J, Rizzi Alberto C, Moura Isabel, Moura José J G, Romão Maria J, Brondino Carlos D
REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
J Am Chem Soc. 2009 Jun 17;131(23):7990-8. doi: 10.1021/ja809448r.
Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.
巨大脱硫弧菌的醛氧化还原酶（DgAOR）是黄嘌呤氧化酶（XO）家族的单核钼酶成员，可催化醛氧化为羧酸。XO家族酶中的钼位点呈现出扭曲的四方锥几何结构，其中两个配体，一个羟基/水分子（催化不稳定位点）和一个硫醇配体，已被证明对催化至关重要。我们在此报告了DgAOR与抑制剂氰化物、乙二醇、甘油和亚砷酸盐的稳态动力学研究，以及该酶与两种醇反应后的晶体学和电子顺磁共振（EPR）研究。与XO家族其他成员的情况不同，氰化物、乙二醇和甘油是DgAOR的可逆抑制剂。氰化物和单晶制备样品的动力学数据证实，DgAOR催化不需要硫醇配体，并证实活性酶中钼原子配位球中不存在该配体。向连二亚硫酸盐还原的DgAOR中添加乙二醇和甘油会产生菱形的Mo(V) EPR信号，表明活性酶近乎四方锥的配位在醇抑制时发生扭曲。这与乙二醇和甘油抑制的酶的X射线结构一致，其中催化不稳定的OH/OH(2)配体丢失，两种醇均以η(2)方式配位钼位点。这两种加合物呈现出钼与醇部分的一个碳原子之间的直接相互作用，这构成了生物系统中这种键的首个结构证据。
