STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES

多组分细菌单加氧酶的结构研究

• 批准号: 7955153
• 负责人: Stephen J. Lippard
• 金额: $ 0.21万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955153
• 关键词: Characteristics; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Set; Enzymes; Family; Funding; Grant; Hydrocarbons; Institution; Metals; Methane hydroxylase; Mixed Function Oxygenases; Phenol 2-monooxygenase; Protein Binding; Pseudomonas; Publications; Reporting; Research; Research Personnel; Resolution; Resources; Role; Site; Site-Directed Mutagenesis; Source; Structure; United States National Institutes of Health; Work; carboxylate; dimer; divalent metal; genetic regulatory protein; improved; mutant; reconstitution; structural biology; toluene 2-xylene monooxygenase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial multicomponent monooxygenases are a family of enzymes that utilize a carboxylate-bridged diiron center to hydroxyalate a variety of hydrocarbon substrates. Essential for this activity is the formation of a complex between the hydroxylase and regulatory protein. The structure of the Pseudomonas sp. OX1 phenol hydroxylase, PHH-PHM complex was determined to 2.3 ¿ resolution. The regulatory protein binds on helices A, E, and F of the hydroxylase alpha subunit at the dimer interface 12 ¿ above the diiron center. Although the metal center resembled the structure of mixed-valent methane monooxygenase, significant structural changes were observed in helices E and F that were different than the configuration typically observed in the uncomplexed form of the hydroxylase. These changes have implications for substrate activation and substrate selectivity, the significance of which is currently being explored. Since determining this structure, structural work has focused on obtaining higher resolution data on the PHH-PHM complex with improved regulatory protein occupancy, and crystallographic characterization of various mutants of PHH and toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1. Higher resolution and structures of the complex with improved regulatory protein occupancy could confirm mechanistic conclusions and speculations about the structural effects of regulatory protein binding proposed upon analysis of the initial PHH-PHM data. Similarly, a structure of the hydroxylase in the absence of the regulatory protein may reveal critical, mechanistically relevant characteristics of the hydroxylase structure while also providing a platform to further investigate the structural effects of regulatory protein binding on the hydroxylase through comparison with the PHH-PHM structure. Diffraction data from these projects are only preliminary and require improvement and analysis before they may be reported. In effort to obtain a diferric or analogous structure of PHH or PHH-PHM, dithionite soaked divalent metal reconstituted and crystals of both species were pursued but not yet successfully obtained such to yield a quality diffraction data set for structure determination. Mechanistic data on mutant forms of ToMOH (T201X) and PHH (N204X) in which conserved residues near the diiron site were varied by site-directed-mutagenesis indicate roles for these residues, so the mutants were crystallized and their diffraction data collected to yield structural information that complements the mechanistic studies. This structural information is presently being analyzed and prepared for publication in conjuction with the relevant mechanistic studies and their results.

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