Characterizing the structure of alkane hydroxylase (AlkB) and related diiron enzy

描述（由申请人提供）：细菌很容易利用金属酶烷烃羟化酶（AlkB）氧化烷烃，因此AlkB的作用代表了自然界对石油泄漏环境影响的最佳防御之一。哺乳动物脂肪酸也可以通过整体膜脂肪酸去饱和酶进行去饱和，其结构与AlkB非常相似。目前尚不清楚为什么AlkB催化氢氧根在烷烃上的加成，而去饱和酶将单键C-C转化为双键C=C。AlkB和可溶性甲烷单加氧酶（sMMO）都使用双铁催化剂催化惰性碳氢键的转化，但AlkB的双铁催化剂主要以氮原子为配体，而sMMO具有富氧配位位点。预测配位环境会影响AlkB的电子结构，从而影响其反应性，但尚未对其电子结构进行表征。这项研究工作的长期目标是了解AlkB的结构如何决定它所影响的化学性质，并利用这些知识加深我们对生物如何选择性地激活分子氧和催化惰性碳氢化合物氧化的理解。一个相关的目标是了解结构非常相似的生物基序是如何在催化烷烃的羟基化或它们的去饱和之间选择的。提出的具体研究的主要目标是获得AlkB的良好衍射晶体，这将使我们能够确定这种酶的三维结构。相关目标是光谱表征基态和活性中间体，并开始探索来自其他微生物的alkb和相关金属酶。这些目标使我们能够验证一个假设，即所有alkb和跨膜去饱和酶的活性位点结构是相同的，但底物结合袋的结构差异控制了底物的选择性和催化剂的反应性。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。AlkB的三维结构将有助于回答许多关于生物学可以用来氧化烷烃的化学基序范围的问题。它还将回答有关石油泄漏环境反应中使用的一些化学过程的问题。

DESCRIPTION (provided by applicant): 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 woul 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.