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1. Design of a minimal di-nickel hydrogenase peptide

   https://doi.org/10.1126/sciadv.abq1990  

   Timm, Jennifer; Pike, Douglas H.; Mancini, Joshua A.; Tyryshkin, Alexei M.; Poudel, Saroj; Siess, Jan A.; Molinaro, Paul M.; McCann, James J.; Waldie, Kate M.; Koder, Ronald L.; et al (March 2023, Science Advances)

   Thorp, Holden (Ed.)

   Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13–amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth. 

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2. De novo design of luciferases using deep learning

   https://doi.org/10.1038/s41586-023-05696-3  

   Yeh, Andy Hsien-Wei; Norn, Christoffer; Kipnis, Yakov; Tischer, Doug; Pellock, Samuel J.; Evans, Declan; Ma, Pengchen; Lee, Gyu Rie; Zhang, Jason Z.; Anishchenko, Ivan; et al (February 2023, Nature)

   Abstract De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds^1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence–structure relationships. Here we describe a deep-learning-based ‘family-wide hallucination’ approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine³and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (k_cat/K_m = 10⁶ M⁻¹ s⁻¹) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes. 

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3. Metal-Ligand Cooperativity in the Soluble Hydrogenase-1 from Pyrococcus Furiosus

   https://doi.org/10.1039/D0SC00628A  

   Vansuch, Gregory E.; Wu, Chang-Hao; Haja, Dominik K.; Blair, Soshawn A.; Chica, Bryant; Johnson, Michael; Adams, Michael W; Dyer, Richard Brian (January 2020, Chemical Science)

   Metal-ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 from Pyrococcus furiosus (Pf SH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Nia3+-C state, which contains a bridging hydride. Instead the tautomeric Nia+-L states were observed. Overall, the results provided insight into complex metal-ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts. 

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4. Advances in Optimizing Enzyme Electrostatic Preorganization

   Hennefarth, M. R.; Alexandrova, A. N. (January 2021, Current opinion in structural biology)

   null (Ed.)

   Utilizing electric fields to catalyze chemical reactions is not a new idea, but in enzymology it undergoes a renaissance, inspired by Warhsel’s concept of electrostatic preorganization. According to this concept, the source of the immense catalytic efficiency of enzymes is the intramolecular electric field that permanently favors the reaction transition state over the reactants. Within enzyme design, computational efforts have fallen short in designing enzymes with natural-like efficacy. The outcome could improve if long-range electrostatics (often omitted in current protocols) would be optimized. Here, we highlight the major developments in methods for analyzing and designing electric fields generated by the protein scaffolds, in order to both better understand how natural enzymes function, and aid artificial enzyme design. 

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5. A semisynthetic, multicofactor artificial metalloenzyme retains independent site activity

   https://doi.org/10.1007/s00775-025-02095-z  

   Wertz, Ashlee_E; Rosenkampff, Ilmari; Ibouanga, Philippe; Huber, Matthias; Hess, Corinna_R; Rüdiger, Olaf; Shafaat, Hannah_S (February 2025, JBIC Journal of Biological Inorganic Chemistry)

   Abstract Native metalloenzymes are unparalleled in their ability to perform efficient small molecule activation reactions, converting simple substrates into complex products. Most of these natural systems possess multiple metallocofactors to facilitate electron transfer or cascade catalysis. While the field of artificial metalloenzymes is growing at a rapid rate, examples of artificial enzymes that leverage two distinct cofactors remain scarce. In this work, we describe a new class of artificial enzymes containing two different metallocofactors, incorporated through bioorthogonal strategies. Nickel-substituted rubredoxin (Ni^Rd), which is a structural and functional mimic of [NiFe] hydrogenases, is used as a scaffold. Incorporation of a synthetic bimetallic inorganic complex based on a macrocyclic biquinazoline ligand (M^MBQ) was accomplished using a novel chelating thioether linker. Neither the structure of the Ni^Rdactive site nor the M^MBQwere altered upon attachment, and each site retained independent redox activity. Electrocatalysis was observed from each site, with the switchability of the system demonstrated through the use of catalytically inert metal centers. This M^MBQ–Ni^Rdplatform offers a new avenue to create multicofactor artificial metalloenzymes in a robust system that can be easily tuned both through modifications to the protein scaffold and the synthetic moiety, with applications for redox catalysis and tandem reactivity. Graphical abstract 

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