Novel tetrapyrrole biosynthetic routes in prokaryotes: Structure and function of enzymes for the biosynthesis of heme d1 in denitrifying bacteria and heme in archaea

环状四吡咯如血红素和叶绿素履行重要的生物功能，并参与有氧和无氧呼吸或光合作用等过程。血红素d1是另一种环状四吡咯，它对细菌的反硝化作用以及全球的氮循环都很重要。硫酸盐还原菌和古生菌中血红素的生物合成途径以及反硝化细菌中血红素d1的生物合成途径是相关的，只是最近才被阐明。在两种途径中发生的一个反应是由西罗血红素脱羧酶催化的西罗血红素的两个乙酸酯基团脱羧为二脱羧西罗血红素的相应甲基。自由基SAM酶AhbD催化西罗血红素依赖性生物合成途径的最后反应，即中间体铁-粪卟啉III的两个丙酸酯基团的氧化脱羧，产生血红素的两个乙烯基。在这个项目中，siroheme脱羧酶和血红素合酶AhbD的催化机制将被调查。在西罗血红素脱羧酶的情况下，存在催化两个脱羧反应中的仅一个或两个的酶的不同同种型。因此，本项目的一个目的是确定各自的氨基酸残基，这是参与底物结合和催化，并确定决定因素的底物和反应特异性的不同siroheme脱羧酶亚型。关于AhbD，将确定脱羧事件的产物，并通过EPR光谱研究酶的自由基机制。另一个目的是确定AhbD的三维结构，特别是与其底物的复合物。

Cyclic tetrapyrroles such as heme and chlorophyll fulfil important biological functions and are involved in processes like aerobic and anaerobic respiration or photosynthesis. Heme d1 is another cyclic tetrapyrrole, which is important for bacterial denitrification and thus for the global nitrogen cycle. The biosynthesis pathways of heme in sulfate-reducing bacteria and archaea and of heme d1 in denitrifying bacteria are related and were elucidated only recently. One reaction occurring in both pathways is the decarboxylation of two acetate groups of siroheme to the corresponding methyl groups of didecarboxysiroheme catalyzed by the enzyme siroheme decarboxylase. The Radical SAM enzyme AhbD catalyzes the last reaction of the siroheme-dependent biosynthesis route to heme, namely the oxidative decarboxylation of two propionate groups of the intermediate iron-coproporphyrin III yielding the two vinyl groups of heme. Within this project, the catalytic mechanisms of the siroheme decarboxylase and of the heme synthase AhbD will be investigated. In the case of siroheme decarboxylase, there are different isoforms of the enzyme that catalyze either only one or both of the two decarboxylation reactions. Therefore, one aim of this project is to identify the respective amino acid residues, which are involved in substrate binding and catalysis, and to identify the determinants responsible for the substrate and reaction specificity of the different siroheme decarboxylase isoforms. Concerning AhbD, the product of the decarboxylation event will be identified and the radical mechanism of the enzyme will be studied by EPR spectroscopy. Another aim is the determination of the three-dimensional structure of AhbD in particular in complex with its substrate.