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Methane Monoxygenase Structure and Function

甲烷单加氧酶的结构和功能

基本信息

批准号：
7815598
负责人：
JOHN D LIPSCOMB
金额：
$ 10.53万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7815598
关键词：
Active Sites Affect Amino Acid Sequence Anabolism Antibiotics Binding Biological Factors C-terminal Catalysis Chemicals Chemistry Chloramphenicol Collaborations Communication Complex Electron Transport Environment Enzymes Experimental Designs Family Family member Hydrolase Hydroxylation Iron Lactamase Lead Link Mass Spectrum Analysis Mediating Metals Methane Methane hydroxylase Methods Mixed Function Oxygenases Optics Oxidases Oxygen Oxygenases Parents Pathway interactions Peptide Sequence Determination Process Production Proteins Protons Reaction Reactive Oxygen Species Regulation Research Proposals Roentgen Rays Role Spectrum Analysis Streptomyces Structure System Zinc Cluster analog base chemical reaction design insight member mutant novel novel diagnostics novel strategies peptide synthase prevent protein folding public health relevance

项目摘要

DESCRIPTION (provided by applicant): The parent proposal sponsors the study of the structure and mechanism of the oxygen-bridged diiron cluster-containing enzyme methane monooxygenase (MMO). Herewe propose a new aim in which the chemistry and regulation of MMO is compared with those of a previously unrecognized diiron monooxygenase family. We have purified the first member of this family (CmlA) from the biosynthetic pathway for chloramphenicol in Streptomyces, where it catalyzes ?-hydroxylation of L-p aminophenylalanine (PAPA). We have shown that both the steady state hydroxylation reaction and the single turnover reaction of the reduced diiron cluster of CmlA with O2 require interaction of CmlA with PAPA covalently loaded on the thiolation domain of a nonribosomal peptide synthetase (NRPS), CmlP. This differs from the O2 activation reaction of the hydroxylase component of MMO (MMOH) that occurs without substrate bound. The amino acid sequence of CmlA shows that it's C-terminal half aligns with the large family of metallo-?-lactamases that usually bind a di-zinc cluster. Nevertheless, metal analysis and EPR and M"ssbauer spectroscopic studies show unequivocally that CmlA binds a diiron cluster. This is the first example of an oxygen activating enzyme using this protein fold. The overall sequence of CmlA aligns with at least 50 uncharacterized enzymes that are part of the biosynthetic pathways for antibiotics and biostatics. We propose to: (i) use truncated CmlA and CmlP constructs to determine the minimal size proteins that can carry out O2 activation and hydroxylation, (ii) Use optical, EPR, and M"ssbauer spectroscopies to characterize the metal center of CmlA and structural perturbations that occur when it binds CmlP analogs, (iii) search for reaction cycle intermediates of CmlA using the single turnover system, and (iv) use diffracting single crystals to determine the X-ray crystal structure of CmlA. The markedly different protein environment and regulatory mechanism for the control of oxygen activation by CmlA should provide an excellent contrast with MMO. We believe that this will allow the roles of the diiron cluster, protein environment, and interactions with other components in diiron oxygenase catalysis to be investigated. The study of the CmlA may also lead to important insights into strategies for the production of novel antibiotics. PUBLIC HEALTH RELEVANCE: We propose to study the O2 activation reaction of the novel dinuclear iron cluster-containing ?-hydroxylase enzyme CmlA from the chloramphenicol biosynthetic pathway of Streptomyces. This enzyme utilzes a protein fold not previously known to support O2 activation. Moreover, the regulation of this process that prevents release of reactive oxygen species is also unique. Protein sequence comparisons suggest that CmlA is the first member of a large family of enzymes involved in antibiotic biosynthesis. The project should provide both fundamental insight into the essential processes of oxygen activation and oxygen incorporation as well as new synthetic strategies for antibiotics and natural products.

说明(申请人提供)：父提案支持对含氧桥连双铁簇酶甲烷单加氧酶(MMO)结构和机理的研究。在这里，我们提出了一个新的目标，将MMO的化学和调节与以前未被发现的双铁单加氧酶家族的化学和调节进行比较。我们从链霉菌中氯霉素的生物合成途径中提纯了这个家族的第一个成员(CmlA)，在那里它催化L-对氨基苯丙氨酸(PAPA)的？-羟基化。我们已经证明，CmlA的稳态羟基化反应和还原的二铁簇与O2的单一翻转反应都需要CmlA与共价负载在非核糖体多肽合成酶(NRPS)硫代化结构域上的PAPA相互作用。这不同于MMO(MMOH)羟基酶成分的O2活化反应，该反应没有底物结合。CmlA的氨基酸序列表明，它的C末端的一半与通常结合双锌簇的金属内酰胺酶大家族一致。然而，金属分析、EPR和穆斯堡尔谱研究明确地表明，CmlA结合了一个双铁原子簇。这是第一个使用这种蛋白质折叠的氧活酶的例子。CmlA的总序列与至少50种未鉴定的酶一致，这些酶是抗生素和生物静止剂生物合成途径的一部分。我们建议：(I)使用截断的CmlA和CmlP结构来确定能够进行O2活化和羟化的最小尺寸的蛋白质；(Ii)使用光学、EPR和穆斯堡尔谱来表征CmlA的金属中心以及当它与CmlP类似物结合时发生的结构扰动；(Iii)使用单一周转系统来搜索CmlA的反应周期中间产物；以及(Iv)使用衍射单晶来确定CmlA的X射线晶体结构。CmlA控制氧激活的蛋白质环境和调控机制明显不同，这与MMO形成了鲜明的对比。我们相信，这将使双铁簇、蛋白质环境以及与其他组分在双铁加氧酶催化中的作用得到研究。对CmlA的研究也可能导致对生产新抗生素的策略的重要见解。公共卫生相关性：我们建议从链霉菌生物合成氯霉素的途径出发，研究含有新型双核铁簇的β-羟基酶CmlA的氧活化反应。这种酶利用一种以前未知的蛋白质折叠来支持O2的激活。此外，对这一过程的调控也是独一无二的，它可以防止活性氧物种的释放。蛋白质序列比较表明，CmlA是参与抗生素生物合成的酶大家族的第一个成员。该项目应提供对氧活化和氧结合的基本过程的基本见解，以及抗生素和天然产品的新合成策略。