Lamm L, Heckmann G, Renz P
Eur J Biochem. 1982 Mar 1;122(3):569-71.
[1-13C]Glycine, [2-13C]glycine, and [15N]glycine were added to fermentations with the strictly anaerobic vitamin B12 producer Eubacterium limosum. The vitamin B12 isolated was almost exclusively labeled in its 5,6-dimethylbenzimidazole moiety. It was methylated and degraded to 1,5,6-trimethylbenzimidazole. The position of the 13C label was determined from the 13 C NMR spectrum using the natural-abundance 13C NMR spectrum of 1,5,6-trimethylbenzimidazole as reference. Thus it was found that C-1 of glycine is incorporated into position 7a, C-2 of glycine into position 3a of the 5,6-dimethylbenzimidazole moiety of vitamin B12. In order to show whether the nitrogen atom of glycine is also incorporated, the vitamin B12 from the experiment with [15N]glycine was degraded to 5,6-dimethylbenzimidazole. The 1H NMR spectrum of this compound exhibited in the region of the C-2 proton a doublet from the 15N-labeled part of the molecules (35%) and a singlet from the unlabeled molecules. This doublet indicates that only one of the two nitrogen atoms of 5,6-dimethylbenzimidazole is labeled. From the incorporation pattern of 13C-labeled glycine it must be deduced that this is N-3 of the 5,6-dimethylbenzimidazole moiety of vitamin B12. These experiments reveal that one molecule of glycine is incorporated into 5,6-dimethylbenzimidazole moiety of vitamin B12 in a regiospecific manner.
将[1-¹³C]甘氨酸、[2-¹³C]甘氨酸和[¹⁵N]甘氨酸添加到严格厌氧的维生素B12产生菌——黏液真杆菌的发酵过程中。分离得到的维生素B12几乎只在其5,6-二甲基苯并咪唑部分被标记。它被甲基化并降解为1,5,6-三甲基苯并咪唑。以1,5,6-三甲基苯并咪唑的天然丰度¹³C NMR谱为参考，从¹³C NMR谱确定¹³C标记的位置。结果发现，甘氨酸的C-1掺入到维生素B12的5,6-二甲基苯并咪唑部分的7a位，甘氨酸的C-2掺入到3a位。为了确定甘氨酸的氮原子是否也被掺入，将用[¹⁵N]甘氨酸进行实验得到的维生素B12降解为5,6-二甲基苯并咪唑。该化合物的¹H NMR谱在C-2质子区域显示出来自分子¹⁵N标记部分的双峰（35%）和来自未标记分子的单峰。这个双峰表明5,6-二甲基苯并咪唑的两个氮原子中只有一个被标记。从¹³C标记甘氨酸的掺入模式可以推断，这是维生素B12的5,6-二甲基苯并咪唑部分的N-3。这些实验表明，一分子甘氨酸以区域特异性方式掺入到维生素B12的5,6-二甲基苯并咪唑部分。
