Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene

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• 作者：Deniz Seyhan ; Dr. Peter Friedrich ; Dr. Maciej Szaleniec ; Dr. Markus Hilberg ; Prof. Dr. Wolfgang Buckel ; Prof. Dr. Bernard T. Golding and Prof. Dr. Johann Heider
• 刊名：Angewandte Chemie International Edition
• 出版年：2016
• 出版时间：September 12, 2016
• 年：2016
• 卷：55
• 期：38
• 页码：11664-11667
• 全文大小：632K
• ISSN：1521-3773

文摘

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.