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Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene |
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| Authors: | Deniz Seyhan Dr. Peter Friedrich Dr. Maciej Szaleniec Dr. Markus Hilberg Prof. Dr. Wolfgang Buckel Prof. Dr. Bernard T. Golding Prof. Dr. Johann Heider |
| Affiliation: | 1. Laboratorium für Mikrobielle Biochemie and Synmikro-Zentrum für synthetische Mikrobiologie, Philipps Universit?t Marburg, Marburg an der Lahn, Germany;2. School of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, UK;3. Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Kraków, Poland;4. Max-Planck-Institut für terrestrische Mikrobiologie, Marburg an der Lahn, Germany |
| Abstract: | Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)‐benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing 2H and 3H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA‐thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate. |
| Keywords: | benzylsuccinate synthase chiral methyl groups glycyl radicals stereochemistry toluene |