Ancestors of Arylmalonate Decarboxylase show increased Activity, Stability and Stereoselectivity

Abstract Bacterial aryl malonate decarboxylase (AMDase) generates a wide spectrum of α-chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. However, extant AMDase from Bordetella bronchiseptica ( Bb AMDase) and related enzymes of the same family have three main limitations: (i) low operational and thermal stability, (ii) limited substrate spectrum regarding the size of the smaller substituent on the α-C-atom, and (iii) low stereoselectivity towards α-alkenyl-α-alkyl malonic acids. To address these limitations, we expanded the structural diversity of the AMDase family by ancestral sequence reconstruction (ASR). The phylogenetic analysis of the decarboxylase revealed conserved structural motifs and key amino acids in the hydrophobic active-site cavity, a catalytic motif crucial for activity and selectivity of the enzyme. The analysis highlighted the natural distribution of amino acid exchanges previously identified via enzyme engineering. AMDase ancestors showed higher stability, activity, and, in one case, also stereoselectivity than Bb AMDase. While a 10 °C higher unfolding temperature of AMDase ancestors is a frequent ASR result, ancestor N131 exhibited an exceptional 294-fold improvement in half-life time and a 1.8-fold increased specific activity (25.1 U·mg⁻¹) toward 2-methyl-2-phenyl malonate in a preparative scale application. Computational analysis attributes this improved stability to a substantial increase in surface Glu side chains, which form a protective hydration shell that enhances solubility and mitigates aggregation. Ancestor N31 formed 2-methyl-but-3-enoic acid from its corresponding malonic acid in excellent optical purity (99.7% eeR ). Finally, the stereoselectivity of the ancestors was completely inverted by switching the catalytic cysteine residues (G74C/C188G).