Roles of protein structure and diiron cluster chemistry in oxygen activation

蛋白质结构和二铁簇化学在氧活化中的作用

• 批准号: 8449094
• 负责人: JOHN D LIPSCOMB
• 金额: $ 29.87万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449094
• 关键词: Active Sites; Address; Amines; Amino Acid Sequence; Amino Acids; Anabolism; Antibiotics; Aromatic Amines; Binding; Biological; Biological Factors; C-terminal; Catalysis; Chemicals; Chemistry; Chloramphenicol; Comparative Study; Complex; Environment; Enzymes; Ethane; Family; Generations; Hydroxylation; Iron; Kinetics; Label; Laboratories; Lactamase; Ligands; Maps; Methane hydroxylase; Mixed Function Oxygenases; Molecular; Molecular Probes; Mutagenesis; Mutate; N-terminal; Nature; Organized by Structure Protein; Oxygen; Oxygenases; Pathway interactions; Peptide Sequence Determination; Pharmaceutical Preparations; Phenylalanine; Play; Process; Production; Proteins; Protocols documentation; Reaction; Regulation; Ribonucleotide Reductase Subunit; Roentgen Rays; Role; Sequence Homology; Spectrum Analysis; Streptomyces; Structure; Surface; System; Techniques; Ursidae Family; Work; antineoplastic antibiotics; base; carboxylate; chemotherapy; insight; member; molecular sieving; novel; overexpression; oxidation; peptide synthase; protein folding; protein structure; quantum; tyrosine radical

DESCRIPTION (provided by applicant): The activation of O2 by diiron cluster-containing enzymes for insertion into biological molecules usually requires a partnership between the cluster, which binds and activates O2, and the protein, which directs and regulates the process in a variety of ways. The well-studied diiron monooxygenases all use carboxylate-rich diiron cluster ligand structures with one His ligand per iron and a 4-helix-bundle protein fold. Moreover, most catalyze reactions such as C-H or aromatic hydroxylation, which are mechanistically related reactions. In order to resolve the roles of the diiron cluster and protein structure in O2 activation, and to further explore the range of chemistry accessible by the diiron cluster oxygenases, new classes of diiron oxygenases are needed that: (i) present the diiron core in a different protein environment, (ii) invoke new core structures, and/or (iii) catalyze novel reactions. While studying the biosynthetic pathway for chloramphenicol from Streptomyces, we found two new diiron enzymes that address these requirements. The first enzyme, CmlA, is the founding member of a class of at least 50 uncharacterized enzymes that catalyze essential ?-hydroxylation of antibiotics, biostatics and chemotherapy agents, as they are synthesized in nonribosomal peptide synthetase (NRPS)-based pathways. Sequence homology, spectroscopy, and product analysis show that CmlA is the first diiron monooxygenase recognized to: (i) bind the diiron cluster in a novel ?-lactamase fold, (ii) catalyze b- hydroxylation, and (iii) incorporae more than one His ligand per Fe. We have shown in preliminary studies using a variety of spectroscopies that the Fe(II)Fe(II) state of CmlA reacts with O2 only when it is complexed with its NRPS (CmlP) covalently loaded with the chloramphenicol precursor L-p-NH2-phenylalanine (PAPA) on its thiolation domain. The second new diiron enzyme, CmlI, catalyzes the final step in chloramphenicol biosynthesis, aromatic amine to nitro conversion. This chemistry has only recently been recognized in the diiron family and is poorly characterized. Preliminary studies show that CmlI uses the 4-helix bundle fold, but, like CmlA, it has more than one His ligand per Fe. Unlike CmlA, it will not catalyze any of the common diiron monooxygenase reactions, and it will accept its substrate either free or bound to CmlP. We propose to structurally characterize CmlA and CmlI with and without their substrates. Efficient single turnover systems for both enzymes will allow transient kinetic techniques to be used to search for and trap reaction cycle intermediates for spectroscopic characterization. Alternative substrates and active site mutagenesis will be used to probe the molecular mechanisms of both enzymes. Spectroscopic labels will be used to reveal the interaction zones with CmlP to investigate the means by which this component regulates O2 binding. Both the intermediates and modes of regulation will be directly compared with those of methane monooxygenase, long studied in this laboratory. This work will bear on fundamental mechanisms of O2 activation and regulation as well as potentially leading to important insights into strategies for the production of novel antibiotics.

描述（由申请人提供）：含二铁簇的酶对O2的激活插入生物分子中通常需要在粘合和激活O2的簇之间建立伙伴关系，该簇与O2的结合和蛋白质（指导和调节过程以多种方式指导和调节过程）。研究良好的二氨基单加氧酶都使用富含羧酸盐的二烷簇配体结构，其每铁的配体和一个4-螺旋 - 荷兰蛋白折叠。而且， 大多数催化反应，例如C-H或芳族羟基化，它们是机械上相关的反应。为了解决二铁群和蛋白质结构在O2激活中的作用，并进一步探索二氨基簇氧化酶可以探索的化学范围，需要新的二氧二氧加氧酶，（i）在不同的蛋白质环境中呈现Diiron Core，在不同的蛋白质环境中，（II）（II）新的核心结构和/或（III III）catyions catyions。在研究链霉菌的氯霉素的生物合成途径时，我们发现了两种解决这些需求的新二铁酶。第一种酶CMLA是至少50种未表征的酶的创始成员，它们催化必不可少的抗生素，生物抑制剂和化学疗法的羟基化，因为它们是在非脱伯蛋白体肽合成酶（NRPS）基于基于的途径中合成的。序列同源性，光谱和产物分析表明，CMLA是识别为：（i）在小说？ - 乳酰胺酶折叠中结合二氧酶的第一个二氧单加氧酶，（ii）催化b-羟基化，而（iii）（iii）浓缩的含量超过每fe。我们在初步研究中使用了多种光谱镜研究表明，CMLA的Fe（II）Fe（II）状态仅在与其NRPS（CMLP）复杂化时与O2的反应，在其Tholiolation domain上与氯霉素前体L-P-P-NH2-苯甲氨酸（PAPA）共价载。 CMLI的第二个新的二铁酶催化了氯霉素生物合成的最后一步，芳香胺至硝基转化。这种化学直到最近才在二龙家族中得到认可，并且特征很差。初步研究表明，CMLI使用了4螺旋束折叠，但是，像CMLA一样，它的配体每fe有多个。与CMLA不同，它不会催化任何常见的二苯基单加氧酶反应，并且它将接受其底物免费或与CMLP结合。我们建议在结构上表征有和没有其底物的CMLA和CMLI。两种酶的有效单周转系统将允许瞬时动力学技术用于搜索和捕获反应周期中间体以进行光谱表征。替代底物和活性位点诱变将用于探测这两种酶的分子机制。光谱标签将用于揭示与CMLP的相互作用区域，以研究该组件调节O2结合的均值。调节的中间体和模式都将直接与该实验室长期研究的甲烷单加氧酶的中间体和模式进行比较。这项工作将基于O2激活和调节的基本机制，并有可能导致对生产新型抗生素的策略的重要见解。