Charakterisierung eines neu entdeckten mitochondrialen molybdäncofaktorabhängigen Enzymsystems

线粒体胺肟还原成分(MARC)是我们实验室于2006年发现的一种含钼酶。除了MARC，人类中只有三种钼酶是已知的。该酶能与细胞色素b5(Cytb5)和NADH细胞色素b5还原酶(B5Red)一起还原N-羟基化合物。几乎所有真核生物基因组都编码两种MARC酶(Marc1和mARC2)，它们的氨基酸序列非常相似。此外，这两种MARC酶都能够与Cytb5和b5RED一起还原N-羟基化化合物。基于我们之前的工作和发表的文献，已经在科学界成功地建立了MARC酶。因此，我们提出的“MARC”这个名称在2011年10月被雨果基因命名委员会(HGNC)采纳。钼辅酶依赖酶系统最初是在研究N-还原药物代谢时发现的。这种参与药物代谢的研究现在已经得到了很好的研究，因此我们的主要目标是探索酶系统的生理功能。我们建议的项目主要是基于我们之前使用分离的重组酶或亚细胞细胞器进行的体外研究。为了更好地评估这种酶系统的生理学相关性，我们未来的研究应该主要使用细胞系统，也应该使用我们的Marc基因敲除小鼠模型。尤其应考虑以下几个方面：(I)是否有一种或两种MARC酶参与了N-还原途径？(2)酶系统是否是阻止突变碱基类似物掺入细胞核酸的细胞保护机制？(Iii)酶系统是否调节L-精氨酸和NO的代谢，从而参与糖尿病/糖尿病肾病？(Iv)线粒体和过氧化物体中的亚细胞定位是否暗示了MARC与活性氧的复杂代谢之间的潜在联系？由于含有MARC的酶系统在含N-羟基基团的药物的代谢中起着核心作用，我们的额外目标是：(V)表征N-羟基苯胺衍生物的还原解毒反应。在这方面，应考虑N-还原酶系统的遗传多态。(Vi)底物化学结构与还原酶活性之间的定量构效关系研究。这一信息以后可能被用于合理开发所谓的“胺磷定”前体药物。我们计划进行的工作是(Vii)从生化光谱角度表征活性中心和功能重要的蛋白质序列基序，以及(Viii)阐明MARC酶的原子结构，这将有助于对这类新酶的总体理解。

The "mitochondrial amidoxime reducing component" (mARC) is a molybdenum-containing enzyme, that was discovered in our labs in 2006. Besides mARC, only three other molybdoenzymes are known in humans. This enzyme is able to reduce N-hydroxylated compounds together with cytochrome b5 (cyt b5) and NADH cytochrome b5 reductase (b5 Red). Almost all eukaryotic genomes encode for two mARC enzymes (mARC1 and mARC2), which are very similar in respect to their amino acid sequence. In addition, both mARC enzymes are able to reduce N-hydroxylated compounds together with cyt b5 and b5 Red.Based on our previous work and publications the enzyme "mARC" is has been successfully established in the scientific world. Thus, the designation "mARC" proposed by us was adopted by the HUGO Gene Nomenclature Committee (HGNC) in October 2011. The molybdenumcofactor-dependend enzyme system was originally discovered during investigations of N-reductive drug metabolism. This involvement in drug metabolism is now well investigated, our primary objective is therefore the exploration of the physiological function of the enzyme system. Our proposed projects are primarily based on our previous in vitro studies using isolated recombinant enzymes or subcellular organelles. In order to better assess the physiological relevance of this enzyme system our future studies should be carried out mainly with cellular systems and also using our mARC-knockout mouse model. In particular the following aspects should be considered: (i) Are one or both mARC enzymes involved in the N-reductive pathway? (ii) Is the enzyme system a cellular protection mechanism that prevents incorporation of mutagenic base analogues into the nucleic acids of the cell? (iii) Does the enzyme system modulates the metabolism of L-arginine and NO and is therefore involved in diabetes/diabetic nephropathy? (iv) Is the subcellular localization in mitochondria and peroxisomes a hint of a potential connection of mARC with the complex metabolism of reactive oxygen species? As the mARC-containing enzyme system plays a central role in the metabolism of drugs with N-hydroxylated functional groups, our additional aims are: (v) The characterization of the reductive detoxification reactions of N-hydroxylated aniline derivatives. In this context genetic polymorphisms of the N-reductive enzyme system should be taken into account. (vi ) The investigation of quantitative structure-activity relationships between the chemical structure of the substrates and the reductase activity. This information could be later applied to the rational development of so-called "amidoxime" prodrugs. Our planned work to (vii) characterize biochemical-spectroscopically both the active center and functionally important protein sequence motifs, and (viii) the elucidation of the atomic structure of mARC enzymes will contribute to the general understanding of this novel class of enzymes.