Biosynthesis of riboflavin
Biosynthesis of riboflavin
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DOI：
10.1111/j.1742-4658.2009.07118.x
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发表时间：
2009-08-01
期刊：
影响因子：
5.4
通讯作者：
Schramek, Nicholas
中科院分区：
文献类型：
--
作者：
Lehmann, Martin;Degen, Simone;Schramek, Nicholas
GTP环化水解酶II催化核黄素生物合成中的第一个专用步骤，并且似乎是重组枯草芽孢杆菌超量生产菌株生产维生素的限制因素。使用易错PCR扩增，我们产生了B的文库。在GTP环化水解酶II结构域中选择性突变的枯草杆菌ribA基因。测量突变蛋白的GTP环化水解酶II与3，4-二羟基-2-丁酮合酶活性的比率。一个突变体命名为构建体E，携带7个点突变，表现出GTP环化水解酶II活性的两倍增加和四倍增加的K-m值与GTP作为底物。使用类似物2-氨基-5-甲酰氨基-6-核糖基氨基-4（3 H）-嘧啶酮5 ′-三磷酸作为底物，突变体显示出约2倍的速率增强和约5倍的K-m值增加。一系列的紫外吸收光谱中获得的停流实验中使用的野生型和突变的酶显示等吸光点指示明显完美的反应，这是类似的发现与GTP环化水解酶II的大肠杆菌。突变体和野生型蛋白质获得的初始爆发速度是相似的。数据表明，存在于构建体E中的突变共同有利于加速反应轨迹中的后期步骤，最可能是产物从酶中的释放。
GTP cyclohydrolase II catalyzes the first dedicated step in the biosynthesis of riboflavin and appears to be a limiting factor for the production of the vitamin by recombinant Bacillus subtilis overproducer strains. Using error-prone PCR amplification, we generated a library of the B. subtilis ribA gene selectively mutated in the GTP cyclohydrolase II domain. The ratio of the GTP cyclohydrolase II to 3,4-dihydroxy-2-butanone synthase activities of the mutant proteins was measured. A mutant designated Construct E, carrying seven point mutations, showed a two-fold increase in GTP cyclohydrolase II activity and a four-fold increase in the K-m value with GTP as the substrate. Using the analog 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate as the substrate, the mutant showed a rate enhancement by a factor of about two and an increase in the K-m value by a factor of about 5. A series of UV absorption spectra obtained in stopped-flow experiments using the wild-type and mutant enzymes revealed isosbestic points indicative of apparently perfect reactions, which were similar to the findings obtained with GTP cyclohydrolase II of Escherichia coli. Initial burst velocities obtained for the mutant and wild-type proteins were similar. The data suggest that the mutations present in Construct E are jointly conducive to the acceleration of a late step in the reaction trajectory, most probably the release of product from the enzyme.