Methane Monoxygenase Structure and Function

描述（由申请人提供）：母提案赞助了含氧桥连二铁簇的酶甲烷单加氧酶（MMO）的结构和机制研究。在此，我们提出了一个新的目标，其中MMO的化学和调节与以前未被识别的二铁单加氧酶家族进行了比较。我们已经从链霉菌中氯霉素的生物合成途径中纯化了这个家族的第一个成员（CmlA），它催化？L-对氨基苯丙氨酸（PAPA）的羟基化。我们已经表明，无论是稳态羟基化反应和单周转反应的减少二铁簇的CmlA与O2需要CmlA的相互作用与PAPA共价加载的硫醇化结构域的非核糖体肽合成酶（NRPS），CmlP。这不同于MMO（MMOH）的羟化酶组分的O2活化反应，其在没有底物结合的情况下发生。CmlA的氨基酸序列表明，它的C-末端的一半与大家族的金属-？-通常结合二锌簇的内酰胺酶。然而，金属分析和EPR和穆斯堡尔光谱研究明确表明，CmlA结合二铁簇。这是使用这种蛋白质折叠的氧活化酶的第一个例子。CmlA的整个序列与至少50种未表征的酶对齐，这些酶是抗生素和生物静力学的生物合成途径的一部分。我们建议：（i）使用截短的CmlA和CmlP构建体来确定可以进行O2活化和羟基化的最小尺寸的蛋白质，（ii）使用光学、EPR和穆斯堡尔光谱来表征CmlA的金属中心和当其结合CmlP类似物时发生的结构扰动，（iii）使用单转换系统来搜索CmlA的反应循环中间体，和（iv）使用衍射单晶来确定CmIA的X射线晶体结构。明显不同的蛋白质环境和调节机制的控制氧激活的CmlA应该提供一个很好的对比MMO。我们相信，这将使二铁簇，蛋白质环境，并与其他组件的相互作用，在二铁加氧酶催化的作用进行调查。对CmlA的研究也可能导致对新型抗生素生产策略的重要见解。 公共卫生相关性：我们建议研究新型双核铁簇合物的O2活化反应。来自链霉菌氯霉素生物合成途径的羟化酶CmlA。这种酶利用以前不知道的蛋白质折叠来支持O2活化。此外，防止活性氧释放的该过程的调节也是独特的。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。该项目将提供对氧活化和氧掺入的基本过程的基本见解，以及抗生素和天然产物的新合成策略。

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.