Figure 4: Synthesis of DHBA isomers via the Claisen condensation reaction.

(a) Formation of 3,4-DHBA; (b) formation of 2,3-DHBA; (c) structures of the deuterium-labelled glycolate, 3,4-DHBA and 2,3-DHBA ammonium adducts detected via HPLC/MS. Encircled and highlighted in red are the α-protons abstracted by the enzyme for the generation of the carbanion responsible for the carbon–carbon bond forming nucleophilic substitution reaction in each case (detailed mechanism depicted in Supplementary Figs S5 and S6). Also highlighted are the respective carbon atoms from each substrate involved in the carbon–carbon bond formation.