On basis of the accepted mechanism of AMDase towards aromatic malonic acids, we formulated two hypotheses to explain the unexpected formation of both enantiomers of 2a by AMDase ICPLLG (Figure ). During the course of our investigation, the experimental results prompted us to formulate a third hypothesis. The first hypothesis assumes two alternative binding modes and a stepwise mechanism. The first binding mode is identical to the decarboxylation of aromatic malonates (Figure , Figure ), in which case the cleaved pro-R carboxylate group is positioned in a hydrophobic pocket, consisting of the same residue positions as the wildtype AMDase, and the pro-S carboxylate is stabilized by the dioxyanion hole (Figure ). In the second binding mode, the substrate is oriented differently, leading to a switched binding of the two carboxylates in the binding pockets (Figure ), which leads to loss of the pro-S carboxylate. After pro-S decarboxylation, protonation of the inversely allocated enediolate intermediate by C74 then forms (R)-2b with retention of the absolute configuration. We propose that both binding modes occur. pro-R decarboxylation would lead to (S)-2b, and pro-S decarboxylation to (R)-2b. Therefore, this hypothesis can be tested by the analysis of the stereochemical pathways. A second hypothesis assumes two protonation pathways and a stepwise pathway with pro-R decarboxylation. A water molecule acts as alternative proton donor (Figure ). This hypothesis is considered to be less likely because structural and computational studies on AMDase did not support the presence of a water molecule in the active site. This hypothesis can be tested by measuring whether only the pro-R carboxylate is cleaved. In the course of this investigation, however, our experimental findings prompted us to formulate a third hypothesis (Figure ). In this, the substrate is bound in two alternative binding modes (Figure ). In contrast to the first hypothesis, the leaving carboxylate group is positioned in a new hydrophobic pocket, formed in part due the introduced mutations (V43I/A125P/V156L/M159L, as well as the L191 residue, already present in wildtype AMDase). Similar to the proposed retention mechanisms, the retained carboxylate is positioned in the dioxyanion hole. The mechanism is enforced concerted with rate-limiting proton transfer (in contrast to the stepwise decarboxylation of arylmalonates, where decarboxylation is rate limiting ). For stereoelectronic reasons, the protonation by C74 and the leaving carboxylate must be at opposite sides of the Ca, leading to inversion of the configuration. Loss of the pro-S carboxylate should form (S)-2b, and loss of the pro-R carboxylate should form (R)-2b. This hypothesis can be tested by analysis of the stereochemical pathways of the decarboxylation and the deuterium kinetic isotope effect ( D KIE) on its rate. In summary, the detection of (at least partial) pro-S decarboxylation would rule out hypothesis 2 (Figure ), whereas hypotheses 1 and 3 could be tested through analysis of the stereochemical pathways in AMDase ICPLLG. To elucidate the mechanistic origin for the unexpectedly low stereoselectivity of this reaction, we wanted to probe the AMDase-catalyzed decarboxylation of isotope-labeled 2-methyl-2-vinyl malonate, together with determination of the kinetic analysis and theoretic QM/MM well-tempered metadynamics calculations.