Martin Philip D, Purcarea Cristina, Zhang Pengfei, Vaishnav Asmita, Sadecki Sharon, Guy-Evans Hedeel I, Evans David R, Edwards Brian F P
Wayne State University School of Medicine, Department of Biochemistry and Molecular Biology, 540 E. Canfield, Detroit, MI 48201, USA.
J Mol Biol. 2005 May 6;348(3):535-47. doi: 10.1016/j.jmb.2005.03.015.
Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C222(1) and C2, were determined at a resolution of 1.7A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted "TIM barrel" domain containing the active site. However, A.aeolicus dihydroorotase has a second, composite domain, which the E.coli enzyme lacks and has only one of the two zinc atoms present in the E.coli enzyme. A.aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E.coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A.aeolicus dihydroorotase can be related to two differences between its structure and that of E.coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a "cysteine switch"; and (2) active site residues that bind the substrate in E.coli dihydroorotase are located in disordered loops in both crystal structures of A.aeolicus dihydroorotase and may function as a disorder-to-order "entropy switch".
二氢乳清酸酶（EC 3.5.2.3）催化氨甲酰天冬氨酸的可逆环化反应，在嘧啶从头合成过程中形成二氢乳清酸。嗜热栖热菌二氢乳清酸酶在两个空间群C222(1)和C2中的X射线结构已确定，分辨率为1.7埃。这些是I型二氢乳清酸酶的首批结构，这类分子包括哺乳动物CAD的二氢乳清酸酶结构域。I型酶比以38 kDa大肠杆菌二氢乳清酸酶为代表的II型酶更古老、更大，为45 kDa。两种二氢乳清酸酶都是金属依赖性水解酶超家族的成员，其成员具有一个包含活性位点的扭曲“TIM桶”结构域。然而，嗜热栖热菌二氢乳清酸酶有第二个复合结构域，大肠杆菌酶没有，且嗜热栖热菌二氢乳清酸酶只有大肠杆菌酶中两个锌原子中的一个。嗜热栖热菌二氢乳清酸酶的独特之处在于，只有与天冬氨酸转氨甲酰酶复合时才表现出显著活性，而大肠杆菌二氢乳清酸酶和CAD二氢乳清酸酶结构域作为游离蛋白时具有活性。嗜热栖热菌二氢乳清酸酶的潜伏性可能与其结构和大肠杆菌二氢乳清酸酶结构之间的两个差异有关：（1）单斜结构有一个与锌结合的新型半胱氨酸配体，该配体阻断活性位点，可能起到“半胱氨酸开关”的作用；（2）在大肠杆菌二氢乳清酸酶中结合底物的活性位点残基位于嗜热栖热菌二氢乳清酸酶的两个晶体结构中的无序环中，可能起到无序到有序的“熵开关”的作用。
