Mechanistic elucidation and stereochemical consequences of alternative binding of alkenyl substrates by engineered arylmalonate decarboxylase

The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenylcarboxylic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase variants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labelled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon, but decarboxylates the corresponding alkenyl malonate with inversion of configuration. Kinetic isotope effect measurements and QM/MM metadynamics calculations suggest that alkenyl malonates adopt an alternative binding mode and undergo decarboxylation via a borderline concerted mechanism instead of a stepwise mechanism. This new pathway changes the stereochemical preference. We exploited this strategy to decarboxylate sterically hindered alkenyl malonates – substrates not converted by wild-type AMDase – with high stereoselectivity. The engineered hydrophobic pocket in (S)-selective AMDase variants expands the substrate scope for synthesizing enantiomerically pure α-aryl and α-alkenyl butanoic acids. This work demonstrates a new approach – a mechanistic change – to engineer the substrate range and stereoselectivity of enzymes.