Romek Katarzyna M, Remaud Gérald S, Silvestre Virginie, Paneth Piotr, Robins Richard J
From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS-University of Nantes UMR6230, F-44322 Nantes, France and the Institute of Applied Radiation Chemistry, Faculty of Chemistry, Łodź University of Technology, ul. Stefana Żeromskiego 116, 90-924 Łódź, Poland.
From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS-University of Nantes UMR6230, F-44322 Nantes, France and.
J Biol Chem. 2016 Aug 5;291(32):16620-9. doi: 10.1074/jbc.M116.734087. Epub 2016 Jun 10.
During the biosynthesis of natural products, isotopic fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs, leading to a non-statistical distribution of isotopes. This can be accessed by isotope ratio monitoring (13)C NMR spectrometry. The solanaceous alkaloids S-(-)-nicotine and hyoscyamine (atropine) are related in having a common intermediate, but downstream enzymatic steps diverge, providing a relevant test case to: (a) elucidate the isotopic affiliation between carbon atoms in the alkaloids and those in the precursors; (b) obtain information about the kinetic isotope effects of as yet undescribed enzymes, thus to make predictions as to their possible mechanism(s). We show that the position-specific (13)C/(12)C ratios in the different moieties of these compounds can satisfactorily be related to their known precursors and to the known kinetic isotope effects of enzymes involved in their biosynthesis, or to similar reaction mechanisms. Thus, the pathway to the common intermediate, N-methyl-Δ(1)-pyrrolinium, is seen to introduce similar isotope distribution patterns in the two alkaloids independent of plant species, whereas the remaining atoms of each target compound, which are of different origins, reflect their specific metabolic ancestry. We further demonstrate that the measured (13)C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified.
在天然产物的生物合成过程中，由于酶对较重或较轻同位素异构体的选择性，会发生同位素分馏。由于分子中只有某些位置参与特定的反应机制，因此会发生位置特异性分馏，导致同位素的非统计分布。这可以通过同位素比率监测（13）C NMR光谱法来实现。茄科生物碱S-（-）-尼古丁和莨菪碱（阿托品）具有共同的中间体，但下游的酶促步骤不同，这为以下方面提供了一个相关的测试案例：（a）阐明生物碱中的碳原子与前体中的碳原子之间的同位素关系；（b）获取有关尚未描述的酶的动力学同位素效应的信息，从而预测其可能的机制。我们表明，这些化合物不同部分中的位置特异性（13）C/（12）C比率可以令人满意地与其已知前体以及参与其生物合成的酶的已知动力学同位素效应或类似反应机制相关联。因此，通向共同中间体N-甲基-Δ（1）-吡咯啉鎓的途径在两种生物碱中引入了相似的同位素分布模式，而与植物物种无关，而每种目标化合物的其余原子来源不同，反映了它们特定的代谢谱系。我们进一步证明，所测得的（13）C分布模式可用于推断仍有待鉴定的酶的反应机制的各个方面。
