Interconnections of metallo enzyme synthesis machineries in Geobacter metallireducens: cofactor assembly and function of W-/FeS-containing BamB

芳香族化合物可以通过非加氧酶途径被厌氧细菌完全降解。在此，大多数单环芳族生长底物转化为中心中间体苯甲酰基-CoA，其在极低的氧化还原电位（E° = -622 mV）下通过还原酶脱芳构化为环状1，5-二烯酰基-CoA。专性厌氧菌，如Fe（III）呼吸模式生物Geophylla metallireducens，使用含有II类苯甲酰辅酶A还原酶的W-辅因子。活性位点组分BamB属于含W的醛：铁氧还蛋白氧化还原酶家族。最初已表征了具有四个[4Fe-4S]簇和催化W-双蝶呤（bisWPT）辅因子的Bam（BC）2配合物。W-原子由来自蝶呤和蛋白质的五个S-原子和尚未鉴定的第六个，可能是氰基配体配位。第6配体介导电子转移从减少W的苯环代表一个前所未有的模式中的Mo/W-辅因子含有酶的催化。该项目的目的是（i）通过一些光谱和结构方法，特别关注关键的第6个W-配体，揭示BamB活性位点的性质和功能;（ii）阐明BamB的不寻常的bisWPT-辅因子的合成和组装，特别是bamL编码的伴侣蛋白的功能;（iii）阐明G.金属还原菌，其能够平行产生Mo-/W-/NiFe/FeS-酶。整个项目是基于与SPP 1927的众多成员和国际专家的密集的方法和主题相关的合作。

Aromatic compounds can be fully degraded by anaerobic bacteria using oxygenase-independent pathways. Here, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-CoA, that is dearomatized by reductases to cyclic 1,5-dienoyl-CoA at an extremely low redox potential (E° = -622 mV). Obligate anaerobes such as the Fe(III) respiring model organism Geobacter metallireducens employ a W-cofactor containing class II benzoyl-CoA reductase. The active site component BamB belongs to the W-containing family of aldehyde:ferredoxin oxidoreductases. A Bam(BC)2 complex with four [4Fe-4S] clusters and a catalytic W-bispterin (bisWPT)-cofactor has initially been characterized. The W-atom is coordinated by five S-atoms from the pterin and the protein and by a yet non-identified 6th, probably cyano-ligand. The 6th ligand mediates electron transfer from the reduced W to the benzene ring representing an unprecedented mode of catalysis among Mo/W-cofactor containing enzymes. The project aims (i) to unravel the nature and function of the BamB active site by a number of spectroscopic and structural methodologies with a special focus on the crucial 6th W-ligand; (ii) to elucidate the synthesis and assembly of the unusual bisWPT-cofactor of BamB, in particular the function of the bamL-encoded chaperone; and (iii) to shed light into the interconnections of metallo enzyme synthesis in G. metallireducens that is capable of producing Mo-/W-/NiFe/FeS-enzymes in parallel. The whole project is based on intensive methodical and topic-related collaborations with numerous members of SPP1927 and international experts.