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Ancestors of Arylmalonate Decarboxylase show increased Activity, Stability and Stereoselectivity

Elske van der PoliD, Jan Georg GerstenbergeriD, Xenia GeorgiadouiD, Gyula HoffkaiD, Lisa-Marie KrammeriD, Klaus Peter SchliepiD, Colin Schür, Shina Caroline Lynn KamerliniD, Selin KaraiD, Robert KouristiD

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Abstract

Bacterial aryl malonate decarboxylase (AMDase) generates a wide spectrum of alpha-chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. However, extant AMDase from Bordetella bronchiseptica (BbAMDase) 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 alpha-C-atom, and (iii) low stereoselectivity towards alpha-alkenyl-alpha-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 BbAMDase. While a 10 degree 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) 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).

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (20 Aug 2026) · CC BY 4.0

Ancestral sequence reconstruction of a bacterial decarboxylase gave enzymes with up to 294-fold longer half-life at 45 °C, higher activity, and 99.7% ee on a vinyl malonate substrate.

The extant Bordetella bronchiseptica AMDase (BbAMDase) has low operational and thermal stability, a narrow substrate range for the smaller α-substituent, and low stereoselectivity toward α-alkenyl malonic acids. The authors argue that ancestral sequences supply a sequence context in which stabilizing or selectivity-enhancing changes are less likely to cost activity than isolated point mutations.

Key results

  1. ShownSeveral ancestors were far more stable than BbAMDase. N131 had a half-life of 381 h at 45 °C against 1.3 h for BbAMDase, and N5 reached 92.6 h. Melting temperature did not predict this ranking.

    Tm by DSF: BbAMDase 55 °C, N164 65 °C, N5 62 °C, N131 62 °C. N131 was 294-fold and N5 72-fold longer-lived at 45 °C. At 30 °C, N5 and N131 kept 68% and 76% activity after 485 h. TTN improved 633-fold for N131. · Analysis of the thermostability of AMDase…

  2. ShownAncestor N131 was faster than BbAMDase on 2-methyl-2-phenyl malonate (2a). In a gram-scale reaction it gave higher specific activity and space-time yield with the same optical purity.

    kcat 34 s^-1, KM 0.3 mM (N131) vs 29 s^-1, 25.5 mM (BbAMDase). Reactor activity 25.1 vs 14.3 U/mg; space-time yield 5.6 vs 3.0 g/L/h; specific productivity 3.4-fold higher; 99.4% vs 99.5% ee; isolated yield 95.8% vs 96.6%. · Enzymatic Activity of Native and Engineered…; Preparative scale application

  3. ShownAncestor N31 decarboxylated 2-methyl-2-vinyl malonate (4a) to the (R)-product with higher enantiopurity than BbAMDase. Isotope-labelling GC-MS indicated that it largely suppresses the pro-S decarboxylation pathway.

    99.7% ee (R)-4b for N31 vs 96.8% for BbAMDase; seven of ten ancestors beat BbAMDase; N5 gave 99.0% ee. Converted to energy differences between the two pathways: 3.9 vs 2.5 kcal/mol. · Analysis of the stereoselectivity of AMDase…; Mechanistic study using pseudochiral…

  4. ShownSwapping the catalytic cysteine (G74C/C188G) plus pocket mutations inverted enantiopreference in ancestors. Some (S)-selective N5 variants beat the engineered BbAMDase ICPLLG variant in activity and selectivity, but the tested N131 LCPLLG and N164 ICPLVG variants were barely active on 1a.

    On 1a: N5 VCPLLG 14.8 and N5 ICPLLG 21 U/mg vs 7.7 U/mg for BbICPLLG. (S)-2b: ≥99.8% ee (N5 VCPLLG, N131 ICPLLG) vs 89.0% for ICPLLG. For (S)-4b, no ancestor variant exceeded 68.8% ee. · Enzymatic Activity of Native and Engineered…; Analysis of the stereoselectivity of AMDase…

  5. SuggestedSimulations and sequence predictions attribute N131's stability to extra surface Glu residues that form an ordered hydration shell. No experimental test of this mechanism, for example by mutating those residues, is reported.

    N131 has a 6% increase in Glu (14 additional negative residues). Instability index 42.2 vs 44.4; SCMTPP score 421.8 vs 404.7. In 10 x 0.5 µs MD runs at 300 and 360 K, N131 showed a higher first hydration peak and more hydrogen bonds and hydrophobic contacts. · Molecular Dynamics Simulations and Analysis; Molecular Dynamics Simulations and Analysis

Methods, data and reproducibility

Maximum-likelihood phylogeny of AMDase sequences rooted with maleate isomerase; marginal ASR of ten nodes, expressed in E. coli and tested for Tm, half-life, kinetics, substrate scope and enantioselectivity by chiral GC. Stability was probed by sequence predictors and Chai-1 models with Amber MD.

  • AMDase sequences plus maleate cis-trans isomerase outgroup used for tree building (HMM built on 136 AMDase sequences) · bacterial · n = 226 sequences (222 AMDase + 4 MI)
  • Ten reconstructed ancestral AMDases (N5, N10, N31, N87, N107, N109, N131, N151, N164, N200) characterized biochemically · bacterial (reconstructed, expressed in E. coli BL21 DE3) · n = 10 ancestors
  • MD simulations of BbAMDase and ancestor N131 at 300 K and 360 K · n = 10 x 0.5 µs replicates per system · Zenodo DOI 10.5281/zenodo.21894315
Methods & tools
phangorn, T-COFFEE, Mafft, MEGAX, Chai-1, Amber22 (pmemd), AmberTools22 (CPPTRAJ), ProtParam, SCMTPP, Protein-Sol, APBS, MDAnalysis
Reagents & environment
phangorn 2.12.1, T-COFFEE 11.00, Amber22, AmberTools22, ff19SB force field with OPC water, PROPKA 3.1, Chimera 1.19.
Assay conditions & compute
GPU-accelerated Amber22 pmemd; 10 x 0.5 µs independent replicates per system (10 µs total over all systems).
Reproducibility gaps
No code repository is stated for the in-house R scripts. Raw simulation data is deposited on Zenodo, given only as a DOI.
Limitations (4)
  • Ancestral nodes could not be reliably predicted from melting temperature, and picking nodes without experimental data on extant enzymes remains a significant limitation. Some ancestors (N87, N151) were poorly active. · stated by the authors
  • Ancestors kept the steric limits of BbAMDase on the smaller α-substituent: low activity on cyclic 6a and only traces of 5b. The (S)-selective N131 LCPLLG and N164 ICPLVG variants were barely active. · stated by the authors
  • The hydration-shell mechanism rests on predictors and MD of a single ancestor (N131) against wild type. No mutational test of the surface Glu residues is reported. · noted in this summary
  • Many activity and selectivity comparisons used cell-free extract rather than purified enzyme, and the improved stereoselectivity of N31 is one ancestor on one substrate. · noted in this summary

Read this if you work on biocatalyst stabilization or ancestral sequence reconstruction and want a worked case with quantified gains in half-life, activity and enantioselectivity.

Citations
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