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不依赖加氧酶的途径被厌氧细菌完全降解。在这里，大多数单环芳香族生长底物被转化为中心中间体苯甲酰辅酶A，该中间体在极低的氧化还原电位(E°=-622 mV)下被还原酶脱芳构化为环1，5-二烯酰辅酶A。专性厌氧菌，如铁(III)呼吸模式生物，金属还原地杆菌利用含有第二类苯甲酰-辅酶A还原酶的W辅因子。活性部位组分BAMB属于含W的铁氧还蛋白氧化还原酶家族。初步表征了一个具有四个[4Fe-4S]簇合物的BAm(BC)_2配合物和一个催化的W-双蝶呤(双WPT)辅因子。W原子由来自蝶呤蛋白和蛋白质的五个S原子和一个尚未鉴定的第六个氰基配体配位。第6个配体介导电子从还原的W到苯环的转移，代表了一种前所未有的包含Mo/W辅因子的酶之间的催化模式。该项目的目的是(I)通过一些光谱和结构方法来揭示BAMB活性中心的性质和功能，特别是关键的第6个W-配体；(Ii)阐明BAMB的不寻常的双WPT辅助因子的合成和组装，特别是BAML编码的伴侣的功能；以及(Iii)揭示金属还原革兰氏菌中能够平行产生Mo/W-/NiFe/FeS酶的金属酶合成的相互联系。整个项目基于与SPP1927众多成员和国际专家的密集、有条不紊和与主题相关的合作。

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.