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-alkenyl alkanoic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (<i>S</i>)-selective AMDase mutants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labeled, 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. The critical Cys residue adds a proton from the same face of the substrate as the leaving carboxylate. Interestingly, the same mutant decarboxylates the corresponding alkenyl malonate with inversion of configuration, i.e., with protonation from the opposite face. 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 (<i>S</i>)-selective AMDase mutants 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.