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

表征烷烃羟化酶 (ALKB) 和相关狄铁酶的结构

• 批准号: 9092672
• 负责人: RACHEL Narehood AUSTIN
• 金额: $ 22.15万
• 依托单位: BARNARD 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 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9092672
• 关键词: Characterizing; structure; alkane; hydroxylase; ALKB

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.

项目概要 细菌利用金属酶烷烃羟化酶 (AlkB) 轻松氧化烷烃，从而发挥 AlkB 是自然界抵御石油泄漏对环境影响的最佳防御手段之一。 脂肪酸也可以通过结构与 AlkB 非常相似的膜内脂肪酸去饱和酶去饱和。 目前尚不清楚为什么 AlkB 会催化 OH 基团与烷烃的加成，而去饱和酶则将其转化为烷烃。 单 C-C 键到双 C=C 键 AlkB 和可溶性甲烷单加氧酶 (sMMO) 均催化该反应。 使用二铁催化剂进行惰性 C-H 键转化，但 AlkB 的二铁催化剂主要是氮 原子作为配体，而 sMMO 具有富氧配位位点，预计配位环境。 影响电子结构并因此影响反应性，但 AlkB 的电子结构尚未被研究 这项研究工作的长期目标是了解 AlkB 的结构。 确定它影响的化学反应，并利用这些知识加深我们对生物学如何影响的理解 选择性激活分子氧并催化惰性碳氢化合物的氧化。 目的是了解结构上非常相似的生物基序如何在催化 烷烃的羟基化或其去饱和度提出的具体研究的主要目标是获得a。 AlkB 的良好衍射晶体将使我们能够确定这种酶的三维结构。 相关目标是对基态和反应中间体进行光谱表征，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结构相同 但底物结合袋的结构差异控制着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。 AlkB 的维度结构将有助于回答有关化学基序范围的许多问题 生物学可以用来氧化烷烃，它也可以回答一些化学过程的问题。 用于石油泄漏的环境响应。

项目成果

Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells.

全细胞中非血红素二铁羟化酶的反应机制。

• 发表时间: 2005-10
• 作者: Bertrand, Erin;Sakai, Ryo;Rozhkova;Moe, Luke;Fox, Brian G;Groves, John T;Austin, Rachel N
• 通讯作者: Austin, Rachel N

Microbial Cell Factories Heterologous Expression of Lcc1 Gene from Trametes Trogii in Pichia Pastoris and Characterization of the Recombinant Enzyme

栓菌 Lcc1 基因在毕赤酵母中的微生物细胞工厂异源表达及重组酶的表征

• DOI: 10.1016/j.biortech.2015.03.149
• 发表时间: 2015-08-01
• 期刊: Bioresource technology
• 影响因子: 11.4
• 作者: Maria Chiara Colao;S. Lupino;A. Garzillo;V. Buonocore;M. Ruzzi
• 通讯作者: M. Ruzzi

Profiling mechanisms of alkane hydroxylase activity in vivo using the diagnostic substrate norcarane.

使用诊断底物降卡烷分析体内烷烃羟化酶活性的机制。

• 发表时间: 2007-02
• 作者: Rozhkova;Chae, Jong;Zylstra, Gerben J;Bertrand, Erin M;Alexander;Deng, Dayi;Moe, Luke A;van Beilen, Jan B;Danahy, Michael;Groves, John T;Austin, Rachel N
• 通讯作者: Austin, Rachel N

Metals in marine biochemistry.

海洋生物化学中的金属。

• 发表时间: 2014-06
• 作者: Austin, Rachel Narehood;Saito, Mak A
• 通讯作者: Saito, Mak A

Cage escape competes with geminate recombination during alkane hydroxylation by the diiron oxygenase AlkB.

在二铁加氧酶 AlkB 的烷烃羟基化过程中，笼逃逸与成对重组竞争。

• 发表时间: 2008
• 作者: Austin, Rachel N;Luddy, Kate;Erickson, Karla;Pender;Bertrand, Erin;Deng, Dayi;Buzdygon, Ryan S;van Beilen, Jan B;Groves, John T
• 通讯作者: Groves, John T