Richter G, Kelly M, Krieger C, Yu Y, Bermel W, Karlsson G, Bacher A, Oschkinat H
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
Eur J Biochem. 1999 Apr;261(1):57-65. doi: 10.1046/j.1432-1327.1999.00211.x.
3,4-Dihydroxy-2-butanone 4-phosphate synthase catalyses the release of C-4 from the substrate, ribulose phosphate, via a complex series of rearrangement reactions. The cognate ribB gene of Escherichia coli was hyperexpressed in a recombinant E. coli strain. The protein was shown to be a 46-kDa homodimer by hydrodynamic analysis. A variety of protein samples labelled with different grades of 13C, 15N and 2H, i.e. one with 100% 2H and 15N, one with 75% 2H, 99% 13C, 15N, and one with 100% 2H, 99% 13C,15N were prepared. Despite the large molecular size, 2- and 3-dimensional NMR spectra of reasonable quality were obtained. Attempts at the assignment of individual 13C, 15N and 1H signals show, in principle, the feasibility of structure determination. The number of NMR signals shows unequivocally that the homodimeric protein obeys strict C2 symmetry.
3,4-二羟基-2-丁酮4-磷酸合酶通过一系列复杂的重排反应催化从底物磷酸核酮糖中释放出C-4。大肠杆菌的同源ribB基因在重组大肠杆菌菌株中过表达。通过流体动力学分析表明该蛋白质是一种46 kDa的同型二聚体。制备了多种用不同等级的13C、15N和2H标记的蛋白质样品，即一种含有100% 2H和15N，一种含有75% 2H、99% 13C、15N，还有一种含有100% 2H、99% 13C、15N。尽管分子尺寸较大，但仍获得了质量合理的二维和三维核磁共振谱。对单个13C、15N和1H信号进行归属的尝试原则上表明了结构测定的可行性。核磁共振信号的数量明确显示同型二聚体蛋白质遵循严格的C2对称性。
