We then investigated the decarboxylation of pro-R-13 C-2a by wildtype AMDase and AMDase ICPLLG. For the analysis of the mass of the product by chiral GC-MS, we analyzed the signal at m/z 85 (M-15). pro-S decarboxylation of (R)-13 C-2a led to loss of the unlabeled carboxylate and formed 1-13 C-2b. pro-R decarboxylation cleaved the 13 C-labeled carboxylate and formed 1-12 C-2b. However, due to the natural abundance of 13 C, 1-12 C-2b we expect that 5% of the molecules contain this isotope. AMDase ICPLLG catalyzed the decarboxylation of pro-R-labeled 2-methyl-2-vinyl malonic acid forming (S)-2b with an ee of 69% (S). In chiral GC-MS analysis, the peak of (S)-2b exhibited a mass ratio of m/z 86:85 = 97:3, indicating loss of the unlabeled pro-S carboxylate. To our knowledge, this is the first time that pro-S decarboxylation has been observed for arylmalonate decarboxylase. It indicates a new, hitherto unknown stereochemical pathway for this enzyme that has not been observed with aromatic substrates and is not compatible with hypothesis 2 (Figure ). Strong tailing of the larger (S)-peak in the chiral GC/MS led to an overlap with the (R)-peak, thus preventing clear analysis of the mass signal of the (R)-enantiomer (Figure ). Nevertheless, despite the overlap, a clear excess of the signal at m/z 85 beyond the natural isotope distribution could be observed in the peak of the (R)-enantiomer. Two variants of AMDase ICPLLG having additional substitutions showed also a preference for pro-Sdecarboxylation and had altered selectivity (Figure , S28-S30). AMDase ICPLLG I43L had a higher selectivity (90% ee), whereas AMDase ICPLLG G190 had lower selectivity (52% ee).