DOI: 10.5281/zenodo.18777210
title: Supplementary Data for "MPNN-guided redesign of PET hydrolases with enhanced catalytic activity below the PET glass transition temperature"

description: This Supplementary Dataset is provided as a compressed ZIP file, which contains the following folders:



CD_25C: Raw data of the circular dichroism (CD) measurements at 25°C between 190 and 250 nm for PHL7 and ProteinMPNN and LigandMPNN redesigns, performed on a Chirascan Q100 CD spectrometer (Applied Photophysics Limited, UK) equipped with a Peltier thermostatic system under constant nitrogen flux with a 0.1 cm quartz cuvette. The curves correspond to the Molar Residual Ellipcitity (MRE), expressed in deg·cm²·dmol⁻¹·res⁻¹.

DSF_2mL: Raw data of the differential scanning fluorimetry (DSF) measurements between 25°C and 99°C at a rate of 0.03°C/s for PHL7 and ProteinMPNN and LigandMPNN redesigns purified from 2 mL E. coli cell cultures, using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, USA) on the QuantStudio3 instrument (Applied Biosystems, USA). Measurements are expressed as -dRFU/dT.

DSF_1L: Raw data of the DSF measurements between 25°C and 99°C at a rate of 0.01°C/s for PHL7 and active ProteinMPNN and LigandMPNN redesigns D5 and D11 purified from 1 L E. coli cell cultures, carried out using SYPRO Orange 5000X (Thermo Fisher Scientific, USA) on the QuantStudio3 instrument (Applied Biosystems, USA). Measurements are expressed as -dRFU/dT.

HPLC_products: Raw data of the HPLC elution profiles of calibration curves and product quantification after PET 24 h microparticle degradation assays using PHL7 and redesigns D5 D11, performed using a Jasco LC-4000 HPLC with autosampler (Jasco Inc., Japan) equipped with a C18 column (Eurosper II 100-5; 150 × 2 mm with pre-column, Knauer Wissenschaftliche Geräte GmbH, Germany) at a flow rate of 1.2 ml/min. The mobile phase consisted of 19:81% acetonitrile:formic acid (0.1% v/v). The sample was diluted 20 times and 10 µL of the dilution were injected, detecting the separation of all products at a wavelength of 260 nm. Measurements are expressed in absorbance units (AU)



SupplData_CalibrationCurve.csv: Raw data of the calibration curves of mixtures of TPA, MHET, and BHET ranging from 0.5-10 mg/L of each product. Commercially available TPA (Sigma-Aldrich, USA), BHET (Sigma-Aldrich, USA), and MHET (Ambeed, USA) were used as standards.

SupplData_EnzymeDegradationHPLC: Raw data of the product quantification after 24 h of degradation of PET microparticles by PHL7, D5 and D11 in buffer composed by 1M K<sub>2</sub>HPO<sub>4</sub> titrated with HCl to pH 8.0, 200 mM NaCl.

Enzyme_Degradation_All_Replicates: Area under the curve (AUC) analysis for quantification of the products released by PHL7, D5 and D11 after degradation of PET microparticles



PDBs: Input and output three-dimensional structures utilized in different parts of the process of redesigning the sequence of PHL7 using ProteinMPNN and LigandMPNN



RosettaRelax: Folder containing the Rosetta-relaxed structure of chain A of the crystal structure of PHL7 bound to TPA (PDB ID 8BRB) used as input for ProteinMPNN and LigandMPNN, and Rosetta-relaxed structures of the ESMFold predicted structures of active redesigns D5 and D11.

ESMFold: Folder containing the ESMFold-predicted structures of active redesigns D5 and D11 using 12 recycling steps

Boltz-2: Folder containing the best ranked Boltz-2 predicted structures of active redesigns D5 and D11 bound to TPA using default settings and MMseqs2 to retrieve a multiple sequence alignment for the protein structure prediction.