LeMaster D M, Cronan J E
J Biol Chem. 1982 Feb 10;257(3):1224-30.
We have developed two Escherichia coli strains for the production of specifically labeled amino acids suitable for high resolution nuclear magnetic resonance experiments. The 13C atoms from the enriched carbon sources, [1-13C]lactate, [1,4-13C2]succinate, and [1-13C]-acetate, are incorporated into the amino acids producing multilabeled molecules with relatively few instances of adjacent enriched carbons. This greatly simplifies the resultant spectra as compared to the extensively spin-coupled spectra of uniformly enriched samples. No isotopic enrichment was found at carbon positions expected to be unenriched by consideration of the major biosynthetic pathways. Utilizing 90% enriched precursors, most positions were enriched to approximately 85% except for those positions derived from acetate and those affected by the carbon interchange of the pentose phosphate shunt. In both of these cases, the enrichment level fell to 70%. The only result enrichment at the delta-methyl carbon of isoleucine. For the bacterial strain in question, the main pathway of isoleucine biosynthesis appears not to be from threonine but from an alternate precursor, possibly glutamate.
我们已经开发出两种大肠杆菌菌株，用于生产适用于高分辨率核磁共振实验的特定标记氨基酸。来自富集碳源[1-¹³C]乳酸盐、[1,4-¹³C₂]琥珀酸盐和[1-¹³C]乙酸盐的¹³C原子被整合到氨基酸中，产生具有相对较少相邻富集碳实例的多标记分子。与均匀富集样品的广泛自旋耦合光谱相比，这极大地简化了所得光谱。通过考虑主要生物合成途径，在预期未富集的碳位置未发现同位素富集。使用90%富集的前体，除了那些源自乙酸盐的位置和那些受磷酸戊糖途径碳交换影响的位置外，大多数位置富集到约85%。在这两种情况下，富集水平降至70%。唯一的结果是异亮氨酸的δ-甲基碳处有富集。对于所讨论的细菌菌株，异亮氨酸生物合成的主要途径似乎不是来自苏氨酸，而是来自另一种前体，可能是谷氨酸。
