Characterizing the structure of alkane hydroxylase (ALKB) and related Diiron Enzymes

项目摘要 细菌容易利用金属酶烷烃羟化酶（AlkB）氧化烷烃，因此 AlkB代表了自然界对石油泄漏环境影响的最佳防御之一。哺乳动物脂肪 酸也被结构上非常类似于AlkB的整合膜脂肪酸去饱和酶去饱和。 不理解为什么AlkB催化OH基团加成到烷烃上，而去饱和酶转化为 单C-C键到双C=C键。AlkB和可溶性甲烷单加氧酶（sMMO）都催化 使用二铁催化剂转化惰性C-H键，但AlkB具有主要含氮的二铁催化剂 sMMO具有富氧的配位位点，而sMMO具有富氧的配位位点。预测了协调环境 以影响电子结构并因此影响反应性，但AlkB的电子结构尚未被 表征了这项研究工作的长期目标是了解AlkB的结构是如何改变的。 决定了它所影响的化学物质，并利用这些知识来加深我们对生物学如何 选择性地活化分子氧并催化惰性烃的氧化。相关联的 目的是了解结构上非常相似的生物基序如何在催化 烷烃的羟基化或它们的去饱和。提出的具体研究的主要目标是获得一个 衍射良好的AlkB晶体将使我们能够确定这种酶的三维结构。 相关的目标是光谱表征基态和活性中间体，并开始 从其他微生物中探索AlkB和相关金属酶。这些目标使我们能够测试 假设所有AlkB和跨膜去饱和酶的活性位点的结构是相同的 但是底物结合口袋中的结构差异控制底物选择性和催化剂反应性。 如果成功，这项工作将对生物无机化学和环境化学产生影响。一个三- AlkB的三维结构将有助于回答许多关于化学基序范围的问题， 可以用来氧化烷烃。它还能回答一些关于 在石油泄漏的环境反应中使用。

Project Summary Bacteria readily oxidize alkanes utilizing the metalloenzyme alkane hydroxylase (AlkB) and thus the actions of AlkB represent one of nature’s best defenses against the environmental effects of oil spills. Mammalian fatty acids are also desaturated by integral membrane fatty acid desaturases that are structurally quite similar to AlkB. It is not understood why AlkB catalyzes the addition of an OH group to an alkane while desaturases convert single C-C bonds to double C=C bonds. AlkB and soluble methane monooxygenase (sMMO) both catalyze the transformation of inert C-H bonds using a diiron catalyst but AlkB has a diiron catalyst with primarily nitrogen atoms as ligands while sMMO has an oxygen-rich coordination site. The coordination environment is predicted to influence the electronic structure and hence reactivity but the electronic structure of AlkB has not been characterized. The long-term objective of this research effort is to understand how the structure of AlkB determines the chemistry it affects and to use this knowledge to deepen our understanding of how biology selectively activates molecular oxygen and catalyzes the oxidation of inert hydrocarbons. An associated objective is to understand how structurally very similar biological motifs select between catalyzing the hydroxylation of alkanes or their desaturation. The primary goal of the specific research proposed is obtain a well-diffracting crystal of AlkB that will enable us to determine the three-dimensional structure of this enzyme. Related goals are to spectroscopically characterize the ground state and reactive intermediates and to begin explore AlkBs and related metalloenzymes from additional microorganisms. These goals enable us to test the hypothesis that the structures of the active sites of all AlkBs and membrane-spanning desaturases are the same but that structural differences in the substrate binding pocket control substrate selectivity and catalyst reactivity. If successful, this work would have an impact on bioinorganic chemistry and environmental chemistry. A three- dimensional structure of AlkB would help to answer many questions about the range of chemical motifs that biology can use to oxidize alkanes. It would also answer questions about some of the chemical processes utilized in the environmental response to oil spills.