02576nam a2200325Ia 4500 MX-MdCICY 20260521091231.0 CICY B-11023 Quantitative isotopic 13C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin Analytical BioChemistry, 393, p.182-188, 2009 Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in 13C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR)spectrometry at natural abundance (NAQ-NMR)has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ-NMR methodology is sufficiently precise and robust to measure the isotope shifts in the 13C/12C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained ven at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl-CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl-CoA as intermediate in the catalytic pathway. BIOTRANSFORMATION CHAIN-SHORTENING REACTION ISOTOPIC 13C NMR SPECTROMETRY FERULIC ACID REACTION MECHANISMS STREPTOMYCES VANILLIN Botosoa, E.P. Blumenstein, C. Mackenzie, D.A. Silvestre, V. Remaud, G.S. Kwiecien, R.A. Robins, R.J. https://drive.google.com/file/d/1rhG-Gezitxt816h4iBJQnqmBqxDcNFBR/view?usp=drivesdk Para ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx Loc REF1 250602s9999 xx |||||s2 |||| ||und|d 21189 21189 0 0 Loc 0 0 F1 CICY CICY RE 2025-06-25 0 B-11023 2025-06-25 14:06:42 2025-06-25 REF1