The cytoplasmic localization and organization of the riboflavin biosynthetic enzymes in Bacillus subtilis and a possible role of the yet uncharacterized protein RibT in complex (FLAVINOSOME) formation/detoxification

首次在原核细胞中，将在体内分析单个生物合成途径的所有酶的详细定位/组织。例如，计划研究负责枯草芽孢杆菌中FMN/FAD生物合成的酶RibGBAH（T）和RibC的定位/组织。假设是酶复合物（“黄蛋白体”）中的底物通道允许这些辅因子的有效合成。作为表征B细胞质内假设的“黄蛋白体”的第一步。枯草芽孢杆菌中，应使用细胞学报告基因研究Rib-enzymes的定位。此外，应通过采用串联亲和纯化（TAP）方法来确定Rib-enzymes的可能相互作用。在这方面，B的作用。subtilis基因ribT具有突出的兴趣。该基因位于核黄素生物合成操纵子ribGBAHT的3'端，其生物学功能尚不清楚。RibT可能具有N-乙酰基转移酶活性。我们的假设是，RibT介导的Rib-enzymes的乙酰化触发核黄素生物合成蛋白质复合物的形成。总而言之，核黄素途径的酶似乎非常适合研究原核细胞中酶的相互作用。

For the first time in a prokaryotic cell, the detailed localization/organization of all enzymes of a single biosynthetic pathway shall be analyzed in vivo. Exemplarily, it is planned to study the localization/organization of the enzymes RibGBAH(T) and RibC responsible for FMN/FAD biosynthesis in Bacillus subtilis. The hypothesis is that substrate channelling in an enzyme complex (“flavinosome”) allows the efficient synthesis of these cofactors. As a first step towards the characterization of a hypothetical “flavinosome” within the cytoplasm of B. subtilis the localization of the Rib-enzymes shall be investigated using cytological reporters. Furthermore, a possible interaction of the Rib-enzymes shall be determined by employing the tandem affinity purification (TAP) method. In this context the role of the B. subtilis gene ribT is of outstanding interest. This gene is located at the 3’ end of the riboflavin biosynthetic operon ribGBAHT, its biological function is still unknown. RibT possibly has N-acetyltransferase activity. Our hypothesis is, that RibT mediated acetylation of the Rib-enzymes triggers the formation of a riboflavin biosynthetic protein complex. All in all, the enzymes of the riboflavin pathway appear to be ideally suited for studying the interplay of enzymes in prokaryotic cells.