Uncovering hidden catalysis through the systematic discovery of synthetic-inspired active sites in natural proteomes

Ser–His–Asp/Glu catalytic triads are among the most widespread and biologically important catalytic architectures in biology, and the evolution of serine hydrolases has long been viewed as involving the diversification, reuse and remodelling of canonical active-site configurations. In this study, we show that catalytic architectures first conceived through synthetic enzyme design then occur naturally within proteomes, thus revealing that evolution has already explored equivalent catalytic solutions. Using six precisely designed synthetic Ser–His–Asp/Glu catalytic triads as functional probes, we systematically investigated natural proteomes for structurally and functionally analogous configurations. One of the six designed architectures was recurrently identified across diverse protein scaffolds not previously recognized as enzymes. Computational analyses and experimental validation using a series of non-activated ester substrates further confirmed that the substrate binding pockets can adopt catalytic geometries compatible with ester hydrolysis. Our results establish a direct experimental link between synthetic catalysis and natural catalysis, and a hidden layer of catalytic potential with implications for protein multifunctionality and evolutionary insights are revealed. In this study, synthetically designed active sites are demonstrated to occur naturally, which challenges prevailing views of enzymatic evolution and moves beyond the assumption that catalytic solutions are confined to canonical protein regions. All files associated with this dataset can be downloaded directly from the Zenodo repository. Spreadsheet files can be opened with Microsoft Excel, LibreOffice Calc, or compatible software. File names and numbering correspond to the supplementary materials cited in the associated publication. Supplementary Table 1 | Structural features of natural, engineered and architecture-guided PET-hydrolysing proteins analysed in this study. a, Summary of catalytic triad and oxyanion-hole assignments for all proteins analysed in this study. b, Geometric parameters of catalytic Ser–His–Asp/Glu triads. c, Identification and structural evaluation of oxyanion-hole residues. d, Planarity and spatial organisation of catalytic architectures. e, Complete structural dataset and geometric descriptors used for catalytic architecture screening. f, Amino-acid sequences and accession information for all proteins included in the analyses. g, Sequence-based pairwise distance matrix. h, Catalytic-centre based distance matrix. Supplementary Table 3 | UniProt homologues of EfeOm and conservation of the engineered catalytic architecture. The table lists the 1,000 homologous proteins retrieved from UniProt using EfeOm as query. Sequence identity values, amino-acid sequences, conservation of catalytic-triad residues and taxonomic classifications based on the Genome Taxonomy Database (GTDB) are provided for each protein. Sixty homologues retain the complete set of residues corresponding to the engineered Ser–His–Asp/Glu catalytic triad. Supplementary Table 4 | Sequence and expression construct information for native and engineered proteins analysed in this study. This table contains the amino-acid sequences of the seven EfeOm homologues selected for experimental characterization. Predicted signal peptides identified using SignalP-5.0 are highlighted in blue. The table also provides information on synthetic gene constructs cloned into pET-45(+) expression vectors (GenScript Biotech, Rijswijk, The Netherlands), and calculated molecular weights obtained using ExPASy Compute pI/Mw. Supplementary Table 4 is provided as a separate Microsoft Excel (.xlsx) file. Supplementary Data 1 | Raw data for in vitro and in vivo activity assays. a.1, Specific activities (µmol min⁻¹ mg⁻¹) of engineered EfeOm and seven natural EfeO homologues determined against the 96-member ester library under the standard assay conditions described in the Methods. The dataset in a.2 includes absorbance measurements at 550 nm recorded during ester hydrolysis following enzyme incubation at 30 °C for the indicated times. Abs/min values were calculated from the linear region of each kinetic trace and subsequently converted into specific activities as described in the Methods. Reactions were performed in 384-well microplates; the identity and plate position of each ester are provided in the worksheet. Each plate contained biological triplicates for each enzyme assayed against 96 ester substrates, together with a negative control. Related to Extended Data Fig. 1. b, PET-hydrolysing activity of purified enzymes. Datasets include the concentrations of PET degradation products and the corresponding specific activity values obtained after incubation of nPET and pPET with purified enzymes at 30, 40 and 60 °C. Product quantification was performed by HPLC using calibration curves (raw chromatograms are provided in Supplementary Data 2). Data are provided in triplicate, including both raw and processed values (with all applied dilution and concentration factors). Related to Extended Data Table 4. c, PET-hydrolysing activity of whole-cell biocatalysts. Datasets include the concentrations of PET degradation products and the corresponding specific activity values obtained after incubation of nPET with selected microorganisms at 30 °C. Product quantification was performed by HPLC using calibration curves (raw chromatograms are provided in Supplementary Data 2). Data are provided in triplicate, including both raw and processed values (with all applied dilution and concentration factors). Related to Extended Data Table 5. Supplementary Data 1 is provided as a separate Microsoft Excel (.xlsx) file. Supplementary Data 2 | Unprocessed HPLC chromatograms. Files are provided in plain-text (.txt) format as exported from the Varian ProStar software (Varian Inc., Palo Alto, CA, USA). The dataset contains the original HPLC chromatograms used for the quantification of PET hydrolysis products reported in this study. Supplementary Data 3 | Uncropped SDS–PAGE gel image underlying Supplementary Fig. 1. The dataset contains the uncropped scans of the 12% Tris–glycine SDS–polyacrylamide gels used for the SDS–PAGE analysis of the purified EfeO homologues shown in Supplementary Fig. 1.