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.

描述（由申请人提供）：父母提案赞助研究含氧二硫簇含氧酶的结构和机理的研究。在这里，我们提出了一个新的目标，其中将MMO的化学和调节与以前未识别的二氨基单加氧酶家族的化学调节进行了比较。我们已经从链霉菌中的氯霉素的生物合成途径中纯化了该家族的第一个成员（CMLA），其中它催化了L-P氨基苯胺（PAPA）的羟基化。我们已经表明，稳态羟基化反应和CMLA还原二铁簇与O2的单个周转反应都需要CMLA与共价载在非二碱基肽合成酶（NRP）的硫醇化结构域上的papa相互作用，CMLP。这与MMO（MMOH）的羟化酶成分的O2激活反应不同，该反应发生没有底物结合。 CMLA的氨基酸序列表明，C末端半与通常结合Di-Zinc簇的大家族的乳糖 - ？ - 乳糖酶对齐。 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使用单个周转系统，（iv）使用衍射的单晶来确定CMLA的X射线晶体结构。 CMLA控制氧气激活的明显不同的蛋白质环境和调节机制应与MMO形成鲜明对比。我们认为，这将允许研究二烷簇，蛋白质环境以及与二氧氧合酶催化中其他成分的相互作用的作用。对CMLA的研究也可能导致对生产新型抗生素的策略的重要见解。公共卫生相关性：我们建议研究含有链霉菌的氯霉素生物合成途径的新型含二核铁簇的O2激活反应。这种酶效果溶解了以前未知可支持O2激活的蛋白质折叠。此外，对阻止活性氧释放的该过程的调节也是独一无二的。蛋白质序列比较表明CMLA是参与抗生素生物合成的大型酶的第一个成员。该项目应既应该提供对氧气激活和氧掺入基本过程的基本见解，又要提供抗生素和天然产物的新合成策略。