Scheuring J, Kugelbrey K, Weinkauf S, Cushman M, Bacher A, Fischer M
Institut für Organische Chemie und Biochemie and Abteilung Elektronenmikroskopie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany.
J Org Chem. 2001 Jun 1;66(11):3811-9. doi: 10.1021/jo001739u.
The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis. The protein comprises a capsid of 60 beta subunits with lumazine synthase activity and a core of three alpha subunits with riboflavin synthase activity. The beta subunits catalyze the formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2). Complexes of recombinant lumazine synthase (beta(60) capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S- or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by (19)F NMR spectroscopy. Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separated signals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and the enzyme. The signal is sensitive to the replacement of amino acid residues F22 and H88. Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomer epimer A in a haloform-like reaction. The elimination reaction is also catalyzed by F22 mutants. The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhanced reaction rate. From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in a position suited to act as proton acceptor in both the physiological as well as the haloform reaction. A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue.
枯草芽孢杆菌的核黄素合酶/鲁玛嗪合酶复合物催化核黄素生物合成的最后两步。该蛋白质由具有鲁玛嗪合酶活性的60个β亚基组成的衣壳和具有核黄素合酶活性的三个α亚基组成的核心。β亚基催化由5-氨基-6-核糖基氨基-2,4(1H,3H)-嘧啶二酮(1)和3,4-二羟基-2-丁酮4-磷酸(2)形成6,7-二甲基-8-核糖基鲁玛嗪(3)。通过(19)F NMR光谱研究了重组鲁玛嗪合酶(β(60)衣壳)与6-三氟甲基-7-氧代-8-核糖基鲁玛嗪(10)以及7S-或7R-6,7-双三氟甲基-8-核糖基鲁玛嗪水合物(11)的复合物。尽管该蛋白质的分子量约为960 kDa，但仍可获得游离配体和结合配体信号分离的光谱。对于11的差向异构体B和酶，游离配体和结合配体的7-三氟甲基信号之间观察到异常大的8 ppm位移差异。该信号对氨基酸残基F22和H88的取代敏感。鲁玛嗪合酶在类卤仿反应中催化R-非对映异构体差向异构体A的7-三氟甲基基团消除。F22突变体也催化消除反应。H88R突变体对差向异构体B表现出相反的立体选择性，反应速率大大提高。从蛋白质活性位点中差向异构体的模型来看，F22侧链的主要功能似乎是使底物环保持在正确的位置。H88在生理反应和卤仿反应中都适合作为质子受体。在H88R突变体的情况下，提出了一种不同的卤仿反应机制，由7-三氟甲基基团与精氨酸残基的胍基之间的氢键引发。
