Non-equivalence of Hydrogen Transfer from Glucose to the pro-R and pro-S Methylene Positions of Ethanol during Fermentation by Leuconostoc mesenteroides Quantified by 2H NMR at Natural Abundance

Abstract

The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a ij ) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative 2 H NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C f ) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C f -1-R:C f -1-S ratio is 2.1, whereas the C f -3-R:C f -3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH-and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.

A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

“…must be the attenuated and superficial manifestations of isotopic differences within molecules" (18). Only recently, however, has it become possible to access these intramolecular distributions of 13 C in whole molecules through the power of irm-13 C NMR at natural abundance to give direct measurement of position-specific 13 C/ 12 C ratios and to obtain quantification of all individual isotopomers (19). This technique has been exploited in authenticity and in examining differences in fermentation pathways.…”

“…To provide logical guidance as to what tracer experiments might be fruitful, we have used the isotope pattern in the extracted tramadol to deduce a potential biosynthetic pathway. This is the first time (to our knowledge) that positionspecific 13 C/ 12 C ratios have been used to make metabolism-guided deductions in relation to deducing a probable biosynthetic route.…”

“…The information that can be obtained from examining the global isotope deviations (δ 13 C g ) in whole molecules (14,15) by isotope ratio monitoring by mass spectrometry (irm-MS) is inadequate because this technique, in which the target is combusted to CO 2 in an O 2 -rich atmosphere, has the disadvantage of only determining the average distribution of 13 C among the isotopomers (δ 13 C g ‰). This leads to considerable loss of information about isotope fractionation, because not only are heavy isotopes unevenly distributed within a compound, but position-specific isotopic fractionation at natural abundance is defined by the specific reactions taking place during biosynthesis (12,16,17).…”

“…The phenomenon of isotopic fractionation during reactions is well established as a method to characterize reaction mechanisms and help distinguish different (bio)synthetic origins (11)(12)(13). The information that can be obtained from examining the global isotope deviations (δ 13 C g ) in whole molecules (14,15) by isotope ratio monitoring by mass spectrometry (irm-MS) is inadequate because this technique, in which the target is combusted to CO 2 in an O 2 -rich atmosphere, has the disadvantage of only determining the average distribution of 13 C among the isotopomers (δ 13 C g ‰).…”

“…To obtain guidance as to the possible primary precursors and intermediates involved, we have taken a novel approach based on studying the nonstatistical distribution of isotopes within the molecule. For 13 C, the powerful technique of isotope ratio monitoring by 13 C NMR (irm- 13 C NMR) spectrometry at natural abundance (6) makes possible the determination of the nonstatistical distribution of 13 C and the observation of individual 13 C isotopomers, thus the intramolecular distributions of 13 C [δ 13 C i (‰)]. To this can be added the values for the δ 18 O, indicating the probable origins of the oxygen atoms present (7).…”