Unusual enzyme complexes in anaerobic aromatic metabolism of Geobacter metallireducens

在兼性厌氧细菌中，对厌氧芳香族代谢已有广泛的研究。由于总体能量代谢的限制，许多关键的酶过程在厌氧细菌中是不同的。例如，在专性厌氧菌(BamBCDEFGHI-亚单位，W/FeS/SeCys/Fad-辅因子)中，依赖于ATP的、脱芳香的I类苯甲酰辅酶A还原酶(BcrABCD-亚基，FeS-辅因子)被专性厌氧菌(BamBCDEFGHI-亚基，W/FeS/SeCys/Fad-辅因子)中不依赖于ATP的II类BCR所取代。在过去的资助期间，我们从金属还原地质杆菌中分离并鉴定了含有活性位点亚基BamBC的未知W/FeS-簇，BamBC的晶体结构使我们能够对其机制进行初步了解。金属还原革兰氏菌中另一个不常见的酶复合体是对甲酚甲基羟基酶(PCMH，PcmGIJ-亚基)，它催化对甲酚厌氧代谢的第一步。它与其他PCMHs的不同之处在于它在膜上的紧密定位，可能与一个类似细胞色素Bc1的复合体(PcmCDEF-亚基)有关，以及它不寻常的亚基/辅因子组成。在上一次筹资期间，我们已经分离并鉴定了可溶性PmcGIJ组分。在下一个资助期，包括迄今缺失的组分在内的II类BCR和PCMH的整个酶复合体都将被分离出来，并用动力学、光谱和结构方法进行研究。主要目的是深入了解新型多组分酶复合体在厌氧细菌中参与异常能量传递/守恒过程的功能。

The anaerobic aromatic metabolism has extensively been studied in facultatively anaerobic bacteria. Due to limitations in the overall energy metabolism many key enzymatic processes differ in obligately anaerobic bacteria. E.g., the ATP-dependent, dearomatizing class I benzoyl-CoA reductase (BcrABCD-subunits, FeS-cofactors) is replaced by an ATP-independent class II BCR in obligate anaerobes (BamBCDEFGHI-subunits, W/FeS/SeCys/FAD-cofactors). In the last funding period we have isolated and characterized the previsouly unknown W/FeS-cluster containing active site subunits BamBC from Geobacter metallireducens, and the crystal structure of BamBC enabled first insights into the mechanism. Another unusual enzyme complex in G. metallireducens is p-cresol methylhydroxylase (PCMH, PcmGIJ-subunits) catalyzing the first steps in anaerobic p-cresol metabolism. It differs from other PCMHs by its tight location in the membrane, possibly in association with a cytochrome bc1-like complex (PcmCDEF-subunits) and by its unusual subunit/cofactor-composition. In the last funding period we have isolated and characterized the soluble PmcGIJ components. In the next funding period both entire enzyme complexes of the class II BCR and PCMH including the so far missing components will be isolated and studied by kinetic, spectroscopic and structural methodologies. A main objective is to obtain insights into the function of novel multi component enzyme complexes involved in unusual energy transduction/conservation processes in obligately anaerobic bacteria.