Discovery of a Concerted Mechanism in Arylmalonate Decarboxylase Expands the Catalytic Scope for Cofactor-free C-C Bond Cleavage

The cofactor-free arylmalonate decarboxylase (AMDase) has been applied for the synthesis of a large number of α-aryl and α-alkenylcarboxylic acids with outstanding stereoselectivity. We observed that (S)-selective AMDase variant ICPLLG produces a single product enantiomer from 2-methyl-2-phenyl malonate with 99% ee (S), but both product enantiomers from 2-methyl-2-vinyl malonate with 66% ee (S). This prompted us to investigate its mechanism. In the decarboxylation of an isotope-labeled, pseudochiral substrate, we observed that the AMDase variant decarboxylates alkenyl malonates only with inversion of the configuration at the α-C atom. This differs from the decarboxylation of arylmethyl malonates, which proceeds with retention. Further mechanistic investigations with kinetic isotope measurements and QM/MM well-tempered metadynamics calculations suggest an enforced concerted mechanism for the decarboxylation of alkenyl malonic acids, which explains the different stereochemical outcome. The alternative binding mode identified in this study, accounts for the decarboxylation of 2-ethyl-2-vinyl malonate in outstanding stereoselectivity, a substrate that is not converted by wild-type AMDase. Our work sheds light on a new mechanism how enzymes can cleave C-C bonds without support of an organic cofactor. The gained mechanistic understanding will provide guidance for future protein engineering efforts.