NONHEME IRON AND THE BIOLOGICAL OXIDATION OF METHANE
非血红素铁和甲烷的生物氧化
基本信息
- 批准号:6180111
- 负责人:
- 金额:$ 39.04万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1983
- 资助国家:美国
- 起止时间:1983-01-01 至 2002-08-31
- 项目状态:已结题
- 来源:
- 关键词:X ray crystallography active sites analog electron nuclear double resonance spectroscopy electron spin resonance spectroscopy electron transport enzyme activity flavins hydroxylation iron laboratory rabbit methane methane monooxygenase nuclear magnetic resonance spectroscopy oxidation ribonucleotide reductase site directed mutagenesis synthetic protein
项目摘要
The major objective of this research is to elucidate the chemical and
physical properties of soluble methane monooxygenase (sMMO), a system of
three proteins used by methanotrophic bacteria to convert methane and
oxygen selectively to methanol and water. Methanotrophs consume
significant amounts of methane, a greenhouse gas and their sole source of
carbon and energy. These organisms are used in bioremediation of the
environment, for example, to remove chlorinated hydrocarbons from drinking
water supplies. Understanding the principles by which the enzyme system
hydroxylates methane can provide key insights into the development of
synthetic catalysts for achieving this important industrial goal. A
principle component of sMMO is the hydroxylase enzyme (MMOH), which house
two non-heme, carboxylate-bridged diiron centers where reductive
activation of dioxygen takes places, evolving species that ultimately
oxidize methane. Related chemistry occurs at similar cores located in the
small subunit of ribonucleotide reductase (RNR), an enzyme which catalyzes
the first step in DNA biosynthesis and which is a target of anti-tumor and
anti-viral agents. Among the specific aims of this project is to
understand the details of how these non-heme iron centers achieve such
remarkable transformations under physiological conditions. Advanced
methodologies will be applied to trap and determine the structures of
intermediates in the MMOH reaction cycle, including rapid freeze-quench
EPR and ENDOR spectroscopic. and double-mixing stopped-flow experiments,
and to examine the reactivity of time-resolved intermediates with
substrates. In parallel with work on the enzyme, synthetic analogs of the
carboxylate-bridged diiron cores of MMOH and RNR will be prepared to help
understand their active sites and to reproduce steps in their catalytic
cycles. Kinetic and mechanistic experiments will be performed the learn
the factors which control alkane hydroxylation, alkene epoxidation,
tyrosyl radical generation, oxidase and peroxidase activities of the
bridged diiron centers. The other two components of the sMMO system, a
reductase (MMOR) and a small coupling protein (MMOB), form complexes with
MMOH and significantly alter its catalytic activity and redox properties.
Additional goals are to determine the structures of both these proteins by
NMR and X-ray diffraction methods and to investigate the formation of
complexes between all three components by thermodynamic and kinetic
measurements. The optical spectra of the flavin and [2Fe-2S] chromophores
in MMOR will be used to track electron-transfer reactions through the
system. Site-directed mutagenesis studies of all three proteins will be
carried out to identify key amino acid residues postulated to be involved
in complex formation, electron transfer, proton transfer, substrate access
to the diiron center, and the hydroxylation chemistry.
本研究的主要目的是阐明化学和
可溶性甲烷单加氧酶 (sMMO) 的物理特性,这是一个系统
甲烷氧化菌用来转化甲烷的三种蛋白质
氧气选择性地分解甲醇和水。甲烷氧化菌消耗
大量甲烷,一种温室气体,也是其唯一来源
碳和能源。这些生物体用于生物修复
环境,例如,去除饮用水中的氯化碳氢化合物
供水。了解酶系统的原理
羟基化甲烷可以为开发提供重要见解
实现这一重要工业目标的合成催化剂。一个
sMMO 的主要成分是羟化酶 (MMOH),它容纳
两个非血红素、羧酸桥联铁中心,其中还原性
双氧的激活发生,进化出最终的物种
氧化甲烷。相关化学发生在位于相似核心处
核糖核苷酸还原酶 (RNR) 的小亚基,一种催化酶
DNA生物合成的第一步,是抗肿瘤和抗肿瘤药物的靶点
抗病毒剂。该项目的具体目标之一是
了解这些非血红素铁中心如何实现这一目标的细节
生理条件下的显着转变。先进的
将应用方法来捕获和确定结构
MMOH 反应循环中的中间体,包括快速冷冻猝灭
EPR 和 ENDOR 光谱。和双混合停流实验,
并检查时间分辨中间体的反应性
基材。在对酶进行研究的同时,还合成了酶的类似物
MMOH 和 RNR 的羧酸桥联铁核将准备好提供帮助
了解它们的活性位点并重现其催化步骤
循环。将进行动力学和机械实验以学习
控制烷烃羟基化、烯烃环氧化的因素,
酪氨酰自由基的产生、氧化酶和过氧化物酶活性
桥接的狄铁中心。 sMMO 系统的另外两个组件是
还原酶 (MMOR) 和小偶联蛋白 (MMOB) 形成复合物
MMOH 并显着改变其催化活性和氧化还原性质。
其他目标是通过以下方式确定这两种蛋白质的结构:
NMR 和 X 射线衍射方法并研究形成
通过热力学和动力学,所有三个组分之间形成复合物
测量。黄素和 [2Fe-2S] 发色团的光谱
MMOR 将用于通过
系统。所有三种蛋白质的定点诱变研究将
进行以确定所涉及的关键氨基酸残基
复杂形成、电子转移、质子转移、底物访问
到二铁中心,以及羟基化化学。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Stephen J. Lippard其他文献
X-ray structure of a dodecamer duplex containing the major cisplatin d(GpG) intrastrand cross-link
- DOI:
10.1016/0162-0134(95)97300-f - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Patricia M. Takahara;Amy C. Rosenzweig;Christin A. Frederick;Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
High resolution crystal structures of the hydroxylase protein of methane monooxygenase
- DOI:
10.1016/0162-0134(95)97479-a - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Amy C. Rosenzweig;Pär Nordlund;Stephen J. Lippard;Christin A. Frederick - 通讯作者:
Christin A. Frederick
Frank Albert Cotton (1930–2007)
弗兰克·艾伯特·科顿(1930 年至 2007 年)
- DOI:
10.1038/446626a - 发表时间:
2007-04-04 - 期刊:
- 影响因子:48.500
- 作者:
Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
Conjugués de nanoparticule de polynucléotide polyvalente en tant que véhicules de distribution pour un agent chimiothérapique
多核苷酸多价纳米粒子结合物与化学药物分配载体
- DOI:
- 发表时间:
2010 - 期刊:
- 影响因子:0
- 作者:
Chad A. Mirkin;David A. Giljohann;W. Daniel;Stephen J. Lippard;Shanta Dhar - 通讯作者:
Shanta Dhar
Structural, mechanistic, and model studies for methane monooxygenase
- DOI:
10.1016/0162-0134(95)97192-s - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
Stephen J. Lippard的其他文献
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{{ truncateString('Stephen J. Lippard', 18)}}的其他基金
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
8362193 - 财政年份:2011
- 资助金额:
$ 39.04万 - 项目类别:
INVESTIGATIONS OF CISPLATIN-DNA CROSS-LINKS ON NUCLEOSOME CORE PARTICLES
核小体核心颗粒上顺铂-DNA 交联的研究
- 批准号:
8169250 - 财政年份:2010
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
8170154 - 财政年份:2010
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES
多组分细菌单加氧酶的结构研究
- 批准号:
8169251 - 财政年份:2010
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7954158 - 财政年份:2009
- 资助金额:
$ 39.04万 - 项目类别:
CHEMISTRY AND BIOLOGY OF PLATINUM ANTICANCER DRUGS
铂类抗癌药物的化学和生物学
- 批准号:
7955152 - 财政年份:2009
- 资助金额:
$ 39.04万 - 项目类别:
Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons
非血红素二铁中心和碳氢化合物的生物氧化
- 批准号:
7923548 - 财政年份:2009
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7954496 - 财政年份:2009
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES
多组分细菌单加氧酶的结构研究
- 批准号:
7955153 - 财政年份:2009
- 资助金额:
$ 39.04万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7721732 - 财政年份:2008
- 资助金额:
$ 39.04万 - 项目类别:
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