Methane Monoxygenase Structure and Function

描述（由申请人提供）：母提案资助对含氧桥联双铁簇酶甲烷单加氧酶（MMO）的结构和机理进行研究。在此，我们提出了一个新的目标，将MMO的化学和调控与以前未被识别的二铁单加氧酶家族进行比较。我们从链霉菌的氯霉素生物合成途径中纯化了这个家族的第一个成员（CmlA），在那里它催化？L-p氨基苯丙氨酸（PAPA）的羟基化。我们已经证明，无论是稳态羟基化反应，还是CmlA的还原二铁簇与O2的单次转换反应，都需要CmlA与共价负载在非核糖体肽合成酶（NRPS）的硫代结构域上的PAPA相互作用。这与没有底物结合的MMO （MMOH）羟化酶组分的O2活化反应不同。CmlA的氨基酸序列表明，它的c端一半与金属-？通常结合二锌簇的内酰胺酶。然而，金属分析、EPR和M“斯堡尔光谱研究明确表明，CmlA结合了一个双铁簇。这是第一个使用这种蛋白质折叠的氧激活酶的例子。CmlA的总体序列与至少50种未表征的酶一致，这些酶是抗生素和生物静力学生物合成途径的一部分。我们建议：(i)使用截断的CmlA和CmlP结构来确定可以进行O2活化和羟基化的最小尺寸蛋白质，（ii）使用光学，EPR和M ' ssbauer光谱来表征CmlA的金属中心和当它与CmlP类似物结合时发生的结构扰动，（iii）使用单转换系统搜索CmlA的反应周期中间体，（iv）使用衍射单晶来确定CmlA的x射线晶体结构。CmlA控制氧活化的蛋白质环境和调控机制明显不同，这应该与MMO形成了很好的对比。我们相信，这将允许研究双铁簇，蛋白质环境和与其他成分在双铁加氧酶催化中的作用。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。

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.