D'Ordine Robert L, Rydel Timothy J, Storek Michael J, Sturman Eric J, Moshiri Farhad, Bartlett Ryan K, Brown Gregory R, Eilers Robert J, Dart Crystal, Qi Youlin, Flasinski Stanislaw, Franklin Sonya J
Monsanto Company, Chesterfield MO 63017, USA. robert.l.d'
J Mol Biol. 2009 Sep 18;392(2):481-97. doi: 10.1016/j.jmb.2009.07.022. Epub 2009 Jul 15.
Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.
麦草畏（2-甲氧基-3,6-二氯苯甲酸）O-脱甲基酶（DMO）是一个由铁氧化还原蛋白和还原酶组成的三组分系统的末端 Rieske 加氧酶。它催化阔叶除草剂麦草畏的 NADH 依赖性氧化脱甲基反应。DMO 代表了一种 Rieske 非血红素铁加氧酶的首个晶体结构，该酶进行环外单加氧反应，将 O₂ 掺入侧链部分而非环系统中。该结构在晶体学不对称单元中显示出一个三重对称三聚体（α₃），相邻亚基间 Rieske 结构域和非血红素铁位点的排列相似，使得电子传递与其他结构特征明确的 Rieske 加氧酶一致。虽然 Rieske 结构域相似，但在催化结构域中观察到了差异，其序列长度比先前描述的要小，但与具有较大底物的加氧酶相比，具有更大体积的活性位点腔。与两亲性底物一致，活性位点设计为通过观察到的关键极性和疏水相互作用与羧酸盐和芳香环相互作用。DMO 的晶体结构在非血红素铁位点存在和不存在底物（麦草畏）、产物（3,6-二氯水杨酸）以及钴或铁的情况下得到了解析。用钴替代铁揭示了一种不常见的非血红素铁结合模式，被非催化性的 Co²⁺捕获，我们推测，在催化循环过程中，这种模式可能在天然酶中短暂存在。因此，我们展示了四个分辨率范围为 1.95 至 2.2 Å 的 DMO 结构，总体上提供了一个动态酶的快照，其中金属结合和底物结合与非血红素铁和催化位点中观察到的结构变化相关联。
