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

表征烷烃羟化酶 (AlkB) 和相关二铁酶的结构

• 批准号: 8573915
• 负责人: RACHEL Narehood AUSTIN
• 金额: $ 9.63万
• 依托单位: BATES COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8573915
• 关键词: Active Sites; Affect; Alcanivorax; Alkane 1-monooxygenase; Alkanes; Bacteria; Binding; Bioinorganic Chemistry; Biological; Biology; Butane; Carbon Dioxide; Chemicals; Chemistry; Cytochrome P450; Dioxygen; Environment; Enzymes; Fatty Acid Desaturases; Fatty Acids; Flavins; Genes; Goals; Gram-Negative Bacteria; Growth; Human; Hydrocarbons; Hydrogen Bonding; Hydroxylation; Knowledge; Ligands; Membrane; Methane hydroxylase; Mixed Function Oxygenases; Molecular; Nature; Nitrogen; Octanes; Oils; Organism; Oxidants; Oxygen; Particulate; Process; Proteins; Reaction; Research; Site; Structure; System; Testing; Toluene; Work; Xylene; catalyst; desaturase; electronic structure; enzyme structure; innovation; interest; lipid metabolism; metalloenzyme; microbial; microorganism; naphthalene dioxygenase; oxidation; pathogen; preference; public health relevance; response; teacher; three dimensional structure

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.

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油类漏油的环境影响的最佳防御力之一。哺乳动物的脂肪酸也通过与ALKB非常相似的整合性膜脂肪酸去饱和酶进行了饱和。尚不清楚为什么ALKB催化OH基团向烷烃添加，而去饱和酶将单个C-C键转换为双C = C键。 ALKB和可溶性甲烷单加氧酶（SMMO）都使用二铁催化剂催化惰性C-H键的转化，但ALKB具有主要是氮原子作为配体的二铁催化剂，而SMMO具有氧气富含氧的辅助位点。预计协调环境会影响电子结构并因此反应性，但尚未表征ALKB的电子结构。这项研究工作的长期目标是了解ALKB的结构如何决定其影响的化学，并利用这些知识来加深我们对生物学如何选择性地激活分子氧的理解并催化惰性烃的氧化。一个相关的目标是了解结构非常相似的生物学基序如何在催化烷基化或其去饱和度之间进行选择。提出的特定研究的主要目标是获得ALKB的鲜明晶体，这将使我们能够确定该酶的三维结构。相关目标是通过光谱镜来表征基态和反应性中间体，并开始探索其他微生物的ALKB和相关的金属酶。这些目标使我们能够检验以下假设：所有ALKB和跨膜的去饱和酶的活性位点的结构是相同的，但是底物结合袖珍控制底物选择性和催化剂反应性的结构差异。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有关生物学可以用来氧化烷烃的化学基序范围的许多问题。它还将回答有关环境对漏油事件的环境反应中使用的一些化学过程的问题。