Carving out a Glycoside Hydrolase Active Site for Incorporation into a New Protein Scaffold Using Deep Network Hallucination

Enzymes are indispensable biocatalysts for numerous industrial applications, yet stability, selectivity, and restricted substrate recognition present limitations for their use. Despite the importance of enzyme engineering in overcoming these limitations, success is often challenged by the intricate architecture of enzymes derived from natural sources. Recent advances in computational methods have enabled the de novo design of simplified scaffolds with specific functional sites. Such scaffolds may be advantageous as platforms for enzyme engineering. Here, we present a strategy for the de novo design of a simplified scaffold of an endo-α-N-acetylgalactosaminidase active site, a glycoside hydrolase from the GH101 enzyme family. Using a combination of trRosetta hallucination, iterative cycles of deep-learning-based structure prediction, and ProteinMPNN sequence design, we designed proteins with 290 amino acids incorporating the active site while reducing the molecular weight by over 100 kDa compared to the initial endo-α-N-acetylgalactosaminidase. Of 11 tested designs, six were expressed as soluble monomers, displaying similar or increased thermostabilities compared to the natural enzyme. Despite lacking detectable enzymatic activity, the experimentally determined crystal structures of a representative design closely matched the design with a root-mean-square deviation of 1.0 Å, with most catalytically important side chains within 2.0 Å. The results highlight the potential of scaffold hallucination in designing proteins that may serve as a foundation for subsequent enzyme engineering

Specific activities and keratin/casein ratios of the purified wild-type and mutant SAPB enzymes using keratin and casein as substrates.

a<p>Specific activity is defined as units (U) of activity per amount (mg) of protein. 1 U of protease activity was defined as the amount of enzyme that liberated 1 µg tyrosine per min under the optimal temperature and pH values of the respective recombinant enzymes using keratin or casein as a substrate. Proteins were estimated by the Bradford method using the Dc protein assay kit obtained from Bio-Rad Laboratories (Inc., Hercules, CA, USA).</p>b<p>The experiments were conducted three times and ± standard errors are reported.</p>c<p>The relative activity is calculated by taking the specific activity of the wild-type as 1.00.</p><p>Specific activities and keratin/casein ratios of the purified wild-type and mutant SAPB enzymes using keratin and casein as substrates.</p

Physico-chemical characteristics of dyed crusts produced from goat skins.

<p>Values represent means of three samples from three sets of experiments, and ± standard errors are reported.</p><p>Physico-chemical characteristics of dyed crusts produced from goat skins.</p

Hydrolysis curves of keratin treated with purified SAPB enzymes.

<p>The purified proteases used were: rSAPB, SAPB-L31I, SAPB-T33S, SAPB-N99Y, SAPB-L31I/T33S, SAPB-L31I/N99Y, SAPB-T33S/N99Y, and SAPB-L31I/T33S/N99Y. Each point represents the mean (n  = 3) ± standard deviation.</p

Analysis of pollution parameters from the effluent of depilating processes of goat skins.

<p>Results were average of three different sets of experiments.</p><p>Analysis of pollution parameters from the effluent of depilating processes of goat skins.</p

Kinetic parameters of the purified wild-type and mutant SAPB enzymes with selected synthetic peptide substrates.

<p>Assays were performed using the purified proteases in 100 mM buffer containing 2 mM Ca²⁺, and 0.2 mM to 50 mM synthetic peptide substrates (YLV and FAAF) at suitable pH. The samples were incubated for 10 min at suitable temperature. Results are mean values from triplicate experiments. 1 U of protease activity was defined as the amount of enzyme that catalyses the transformation of 1 mM pNA per minute under standard assay conditions.</p><p>Kinetic parameters of the purified wild-type and mutant SAPB enzymes with selected synthetic peptide substrates.</p

Substrate specificities of the wild-type and mutant SAPB enzymes with proteins and synthetic peptides as substrates.

a<p>Values represent means of three replicates, and ± standard errors are reported.</p>b<p>The unit activity of each substrate was determined by measuring absorbance at specified wavelengths as described in Section 2.</p><p>Substrate specificities of the wild-type and mutant SAPB enzymes with proteins and synthetic peptides as substrates.</p

Structural interpretation.

<p>(A) SAPB model showing the positions of the mutated amino-acids. (B) Surface representation of SAPB which the YLV tripeptide synthetic substrate, shown in orange sticks, has docked. Close up views of (C) the catalytic cavity and Leu31, showing a superposition of surface and ribbon representations of the SAPB model, (D) the catalytic cavity and the Ile31 residue in the SAPB-L31I model. (E) Thr33 in the SAPB model showing a superposition of surface and ribbon representations, and (F) Ser33 in the SAPB-T33S model showing a superposition of surface and ribbon representations. (G) Asn99 in the SAPB model showing a superposition of surface and ribbon representations, and (H) Tyr99 in the SAPB-N99Y model showing a superposition of surface and ribbon representations. The mutated residues are shown in yellow sticks and surfaces. These figures were prepared using the PyMol software (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p

Depilation activities of SAPB enzymes on animal hides and a scanning electron micrograph-selected sectional view.

<p>SAPB enzymes were incubated for 8 h at 37°C with goat skin (A), rabbit hair (B), cow hide (C), and sheep wool (D). Every test was carried out with a control without adding enzyme. Magnification and micrographs of 61× (E), 217× (F), and 435× (G) were taken following the treatment of goat skin with rSAPB enzyme-assisted depilation. Samples show a clean pore, indicating complete removal of the hair and root.</p

Crystallization of BmrA extracted with C4C10.

<p>BmrA was extracted and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g005" target="_blank">Figure 5</a>, using C4C10 instead of C4C7 and exchanging it with FC12. (A) The protein, concentrated to 10 mg/ml, and mixed with 1 mM doxorubicin, crystallized after 10 days in 0.2 M KSCN, 20% PEG 3350, and was (B) analyzed at the ESRF beamline ID23EH-2.</p