The enzymatic decarboxylation of a-arylmalonates is constituted by two highly stereoselective steps (Figure ). With the decarboxylation of pseudochiral 13 C-labeled 2-methyl-2-phenyl malonate, Ohta and Miyamoto showed that exclusively the pro-R carboxylate is lost while the pro-S carboxylate is retained (Scheme S1). This was also confirmed by Micklefield and co-workers using a similar pro-R 18 O-labeling strategy. The determination of the 3D-structure of AMDase showed that the pro-S carboxylate is coordinated by several hydrogen bonds in the so-called 'dioxyanion hole', while the pro-R group points into a hydrophobic pocket whose unfavorable interaction with the charged carboxylate causes the breaking of the C-C bond. Protonation of the resulting enediolate by C188 then forms (R)-1b under inversion of the configuration of the Ca. Lind et al. argued for the involvement of a planar enediolate intermediate and a stepwise mechanism through DFT cluster calculations on a truncated active site system. Their calculations show a much more favorable transition state energy for pro-R cleavage than for an alternative pathway via pro-S decarboxylation. The decarboxylation step (although stereoselective itself) leads to a planar enediolate intermediate and does not contribute to the overall stereoselectivity of the enzymatic process. In contrast, the stereoselective outcome of the reaction is determined by the protonation of the enediolate intermediate by a catalytic cysteine: Protonation by C188 in the decarboxylase from Bordetella bronchiseptica leads to exclusive formation of the (R)-product under inversion of the stereocenter (Figure ). Inspired by the structural similarity of AMDase to glutamate racemase, Ohta and Miyamoto created the variant AMDase G74C/C188S, in which the catalytic Cys is placed on the opposite side of the planar enediolate. This variant exclusively forms the (S)-product. Ohta and Miyamoto confirmed with the decarboxylation of isotope-labeled arylmalonates by AMDase C188S/G74C that also the (S)-selective variants still exclusively cleave the pro-R carboxylate. The fact that the decarboxylation of arylmalonates by variants having C74 can proceed under retention of the absolute configuration at the Ca confirms the stepwise mechanism proposed and rules out a concerted mechanism.