STRUCTURAL STUDIES OF REDOX STATES OF MAUG

MAUG 氧化还原态的结构研究

• 批准号: 8362313
• 负责人: CAROLINE MARY WILMOT
• 金额: $ 0.38万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362313
• 关键词: Amino Acids; Anabolism; Complex; Electrons; Environment; Enzymes; Funding; Grant; Heme; Hydrogen Peroxide; Indoles; Ligands; Molecular; Mono-S; National Center for Research Resources; Oxidation-Reduction; Oxygen; Post-Translational Protein Processing; Principal Investigator; Protein Precursors; Radiation; Reaction; Research; Research Infrastructure; Resolution; Resources; Site; Solutions; Source; Structure; Tryptophan; United States National Institutes of Health; Work; cofactor; cost; covalent bond; crosslink; methylamine dehydrogenase; novel; oxidation; polypeptide; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this collaborative project is to structurally characterize different redox states of MauG. MauG is a highly unusual di-heme enzyme that completes the synthesis of the novel amino acid derived catalytic cofactor, tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ is formed by post-translational modification of two Trp residues in the MADH beta polypeptide chain during which two atoms of oxygen are incorporated into the indole ring of betaTrp57 and a covalent bond is formed between the indole rings of betaTrp57 and betaTrp108. The natural substrate for MauG (preMADH) is a 119 kDa protein precursor of MADH with mono-hydroxylated betaTrp57 and no cross-link. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis. It can do this in a H2O2-dependent or O2/reducing equivalents-dependent reaction. The enzyme has been shown to form a unprecedented di-heme bis-Fe(IV) species that is catalytically competent. This intermediate is electronically equivalent to Fe(V), and is composed of an Fe(IV)=O with the second oxidizing equivalent residing on the Fe of the second heme. We have solved the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1 ¿, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The 5-coordinate P-heme that we expect to be the site of Fe(IV)=O formation is 32 ¿ from TTQ. The 6-coordinate E-heme lies equidistant between the P-heme and TTQ, and has a highly unusual His/Tyr axial ligand configuration that we hypothesize is required for stabilizing Fe(IV). Using XAS both in solution and in the crystal, we wish to characterize the molecular environments that can support and stabilize the unusual redox species of MauG, and enable us to make fundamental discoveries about oxygen activation by an unprecedented di-heme bis-Fe(IV) intermediate. This work would be the first XAS study of this unusual enzyme.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 该协作项目的目的是在结构上表征Maug的不同氧化还原状态。 Maug是一种高度不寻常的双 - 血红素酶，可以完成新型氨基酸衍生的催化辅助因子，色氨酸素酮（TTQ），在甲胺酶脱氢酶（MADH）中发现。 TTQ是通过对MADHβ多肽链中两个TRP残基的翻译后修饰形成的，在此期间，将两个氧原子掺入Betatrp57的吲哚环中，并在Betatrp57和Betatrp108的吲哚环之间形成共价键。 Maug（Premadh）的天然底物是MADH的119 kDa蛋白前体，具有单羟基化的betatrp57，无交叉链接。 MAUG催化六电子氧化物以完成TTQ生物合成。它可以在H2O2依赖性或O2/还原等效依赖性反应中执行此操作。酶已显示出形成一种前所未有的双 - 纤维 - FE（IV）物种，具有催化能力。该中间体在电子上等效于Fe（V），由Fe（IV）= O组成，第二个氧化等效物位于第二血红素的Fe上。我们已经解决了与Premadh复合物中MAUG的晶体结构。这已经达到了2.1€的分辨率，这些晶体可以支持催化转换以形成TTQ而不会衍射。我们期望成为Fe（IV）= O形成的位置的5坐标P-血 - 来自TTQ的32。 6坐标e-eme在p - 血红素和TTQ之间等效，并且具有高度不寻常的/tyr轴向配体构型，我们假设稳定Fe（IV）需要我们假设的。在溶液和晶体中都使用XAS，我们希望表征可以支持和稳定Maug的异常氧化还原物种的分子环境，并使我们能够通过前所未有的Di-Heme Bis-Fe（IV）中间体进行有关氧气激活的基本发现。这项工作将是对这种不寻常酶的首次XAS研究。