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Protein engineering is the design and modification of protein structures to optimize their functions or create novel functionalities for applications in biotechnology, medicine or industry. It represents an essential scientific solution for many of the environmental and societal challenges ahead of us, such as polymer degradation. Unlike traditional chemical methods, enzyme-mediated degradation is selective and environmentally friendly and requires milder conditions. Computational methods will play a critical role in developing such solutions by enabling more efficient bioprospecting of natural polymer-degrading enzymes. They provide structural information, generate mechanistic studies, and formulate new hypotheses, facilitating the modeling and modification of these biocatalysts through enzyme engineering. The recent development of pluriZymes constitutes an example, providing a rational mechanism to integrate different biochemical processes into one single enzyme. In this review, we summarize our recent efforts in this line and introduce our early work towards polymer degradation using a pluriZyme-like technology, including our latest development in PET nanoparticle degradation. Moreover, we provide a comprehensive recipe for developing one’s own pluriZyme so that different laboratories can experiment with them and establish new limits. With modest computational resources and with help from this review, your first pluriZyme is one step closer., This study was conducted under the auspices of the FuturEnzyme Project, funded by the European Union’s Horizon 2020 Research and Innovation Programme under the FuturEnzyme Project (Grant Agreement No. 101000327) and by Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) grant PID2019-106370RB-I00 (VG). A.R.-M. is grateful for grant Nos. PRE2020-091825 from the Spanish Ministry of Science and Innovation, and S.R. thanks the Spanish Ministry of Science and Innovation for providing a Ph.D. fellowship (FPU19/00608)., Peer Reviewed, Postprint (published version)

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5\_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5\_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5\_2 enzymes towards galactose-containing saccharides., We are grateful to Pom Geslin-Vinck for her assistance in constructing some of the CgrAlcOx loop variants. This study was supported by (a) the ‘Centre National de la Recherche Scientifique’ (CNRS), (b) the French ‘Agence Nationale de la Recherche’ (ANR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) through the joint ‘Projet de Recherche Collaboratif International’/‘Strategic Partnership Grants for Projects’ program, supporting the project entitled ‘FUNTASTIC—Fungal copper radical oxidases as new biocatalysts for the valorization of biomass carbohydrates and alcohols’ (ANR-17-CE07-0047, NSERC STPGP 493781–16) and (c) the Spanish Ministry of Innovation and Sciences (PID2019-106370RB-I00)., Peer Reviewed, Postprint (published version)

Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function (“X282”). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members., his work was supported by a grant overseen by the French National Research Agency (ANR) (SNOEBORD project, grant number ANR-21-CE43-0025). Computational work was supported by grant PID2019-106370RB-I00 from the Spanish Ministerio de Ciencia e Innovación., Peer Reviewed, Postprint (published version)

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h−1), catalyst performance (up to 84 s−1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity., BT/Biocatalysis

With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active, demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively., Peer Reviewed, Postprint (published version)

Accumulated greenhouse gas emissions are expected to increase from 36.2 Giga-tons (Gt) to 60 Gt over the next three decades. The global surface temperature has increased by¿+¿1.09¿°C since 2001, and might increase by¿+¿2.2¿°C in 2100, +3.6¿°C in 2200 and +4.6¿°C in 2500. These emissions and temperature rises cannot be reduced in their entirety, but they can be lowered by using enzymes. Enzymes are proteins that catalyze biochemical reactions that make life possible since 3.8 billion years ago. Scientists have been able to "domesticate" them in such a way that enzymes, and their engineered variants, are now key players of the circular economy. With a world production of 117 Kilo-tons and a trade of 14.5 Billion-dollars, they have the potential to annually decrease CO2 emissions by 1 to 2.5 Billion-tons (Bt), the carbon demand to synthesise chemicals by 200 Million tons (Mt), the amount of chemicals by 90¿Mt, and the economic losses derived from global warming by 0.5%, while promoting biodiversity and our planet¿s health. Our success to increase these benefits will depend on better integration of enzymatic solutions in different sectors.

The identification or design of biocatalysts to mitigate the accumulation of plastics, including sub-micro- and nano-sized polyethylene terephthalate (nPET), is becoming a global challenge. Here we computationally incorporated two hydrolytic active sites with geometries similar to that of Idionella sakaiensis PET hydrolase, to fragaceatoxin C (FraC), a membrane pore-forming protein. FraCm1/m2 could be assembled into octameric nanopores (7.0 nm high × 1.6–6.0 nm entry), which deconstructed (40 °C, pH 7.0) nPET from GoodFellow, commodities and plastic bottles. FraCm1 and FraCm2 degrade nPET by endo- and exo-type chain scission. While FraCm1 produces bis(2-hydroxyethyl) terephthalate as the main product, FraCm2 yields a high diversity of oligomers and terephthalic acid. Mechanistic and biochemical differences with benchmark PET hydrolases, along with pore and nPET dynamics, suggest that these pore-forming protein catalytic nanoreactors do not deconstruct macro-PET but are promising in nanotechnology for filtering, capturing and breaking down nPET, for example, in wastewater treatment plants. [Figure not available: see fulltext.]. © 2023, The Author(s).