Schott K, Kellermann J, Lottspeich F, Bacher A
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Federal Republic of Germany.
J Biol Chem. 1990 Mar 15;265(8):4204-9.
Bacillus subtilis has two different riboflavin synthases characterized by the subunit structures alpha3 (light enzyme) and alpha3beta60 (heavy enzyme). The light enzyme was purified by a novel procedure with increased yield and excellent reproducibility. The proposed trimer structure was confirmed by cross-linking experiments with dimethyl suberimidate. Fragments of alpha subunits were prepared by cleavage with cyanogen bromide, trypsin, protease Lys-C, and Staphylococcus aureus protease V8, respectively. Sequences were determined by automated liquid or gas phase Edman degradation. The complete sequence (202 amino acids) was established by direct sequencing of the N terminus and sequencing of overlapping peptides. The sequence shows marked internal homology between the NH2-terminal and COOH-terminal half encompassing 26 identical positions and 23 conservative replacements. This suggests that the protomer forms two structurally similar domains. Since it is known that the enzyme has two binding sites per subunit for the substrate 6,7-dimethyl-8-ribityllumazine, it appears likely that each of the homologous protein domains provides one binding site. The stereochemical features of the enzyme mechanism and the structural relation of the alpha trimer to the beta60 capsid of heavy riboflavin synthase suggest that the six domains corresponding to the alpha subunit trimer are related by pseudo 32 symmetry.
枯草芽孢杆菌有两种不同的核黄素合酶，其特征在于亚基结构α3（轻酶）和α3β60（重酶）。通过一种新方法纯化了轻酶，该方法提高了产量并具有出色的重现性。通过用辛二酸二甲酯进行交联实验证实了所提出的三聚体结构。分别用溴化氰、胰蛋白酶、赖氨酸C蛋白酶和金黄色葡萄球菌蛋白酶V8切割制备α亚基片段。通过自动液相或气相埃德曼降解法测定序列。通过N端直接测序和重叠肽测序确定了完整序列（202个氨基酸）。该序列在NH2端和COOH端的一半之间显示出明显的内部同源性，包括26个相同位置和23个保守替换。这表明原体形成两个结构相似的结构域。由于已知该酶每个亚基有两个底物6,7-二甲基-8-核糖基卢马嗪结合位点，因此每个同源蛋白质结构域似乎提供一个结合位点。酶机制的立体化学特征以及α三聚体与重核黄素合酶的β60衣壳的结构关系表明，与α亚基三聚体相对应的六个结构域通过伪32对称性相关。
