title: Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.
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 (2) H and (3) H 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.
authors: Laboratorium für Mikrobielle Biochemie and Synmikro-Zentrum für synthetische Mikrobiologie, Philipps Universität Marburg, 35043, Marburg an der Lahn, Germany.
authors: Deniz
authors: D
authors: Seyhan
authors: School of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
authors: Peter
authors: P
authors: Friedrich
authors: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Science, 30-239, Kraków, Poland.
authors: Maciej
authors: M
authors: Szaleniec
authors: Laboratorium für Mikrobielle Biochemie and Synmikro-Zentrum für synthetische Mikrobiologie, Philipps Universität Marburg, 35043, Marburg an der Lahn, Germany.
authors: Markus
authors: M
authors: Hilberg
authors: Max-Planck-Institut für terrestrische Mikrobiologie, 35043, Marburg an der Lahn, Germany.
authors: Wolfgang
authors: W
authors: Buckel
authors: School of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
authors: Bernard T
authors: BT
authors: Golding
authors: Laboratorium für Mikrobielle Biochemie and Synmikro-Zentrum für synthetische Mikrobiologie, Philipps Universität Marburg, 35043, Marburg an der Lahn, Germany. heider@biologie.uni-marburg.de.
authors: Johann
authors: J
authors: Heider
mesh_terms: D019755:Carbon-Carbon Lyases / Q000378:metabolism*; D005650:Fumarates / Q000737:chemistry; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013386:Succinates / Q000737:chemistry / Q000378:metabolism*; D014050:Toluene / Q000737:chemistry* / Q000378:metabolism; D014316:Tritium / Q000737:chemistry
chemical_list: D005650:Fumarates; D013386:Succinates; D014316:Tritium; C030196:benzylsuccinate; D014050:Toluene; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase
keywords: benzylsuccinate synthase; chiral methyl groups; glycyl radicals; stereochemistry; toluene