Baldwin J E, Adlington R M, Coates J B, Crabbe M J, Crouch N P, Keeping J W, Knight G C, Schofield C J, Ting H H, Vallejo C A
Dyson Perrins Laboratory, University of Oxford, U.K.
Biochem J. 1987 Aug 1;245(3):831-41. doi: 10.1042/bj2450831.
Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.
来自顶头孢霉（产黄顶头孢霉）的脱乙酰氧基头孢菌素C合成酶（扩环酶）经SDS/聚丙烯酰胺凝胶电泳判断已纯化至接近均一。该酶（分子量约40,000）的最适pH约为7.5。它需要2-氧代戊二酸（Km 0.04 mM）、Fe2+和O2作为辅因子，并且抗坏血酸和二硫苏糖醇对最大活性是必需的。在1 mM二硫苏糖醇存在下，它在-70℃可稳定4周以上。活性受到硫醇淬灭试剂N-乙基马来酰亚胺、金属离子螯合试剂邻二氮菲和NH4HCO3的抑制。高度纯化的酶还表现出脱乙酰氧基头孢菌素C羟化酶（脱乙酰头孢菌素C合成酶）活性，表明扩环酶和羟化酶活性都是单一蛋白质的特性。这些活性不能通过离子交换、染料配体、凝胶过滤或疏水色谱分离。合成了青霉素N的β-亚砜和3β-亚甲基羟基类似物以测试其作为环扩反应潜在中间体的可能性，两种化合物都不是该酶的底物。一种其中青霉素N的3β-甲基和2-氢原子被环丙烷环取代的合成类似物不是底物，但却是该酶的可逆抑制剂。
