• Design, Optimization and Characterization of a de novo Gold Hydroaminase
  Enzymes Enzymes
  Elinor Morris, Boris Lozhkin, Jan Uhrhan, Indrek Kalvet, Sophie Basler, Roman P. Jakob, David Baker, Thomas R. Ward. ACS Catalysis, 2026 | doi:10.1021/acscatal.5c08100
  
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Lab-Led

• Computational design of metallohydrolases
  Enzymes Enzymes
  Kim D, Woodbury SM, Ahern W, Tischer D, Kang A, Joyce E, Bera AK, Hanikel N, Salike S, Krishna R, Yim J, Pellock SJ, Lauko A, Kalvet I, Hilvert D, Baker D.
  Nature, 2026 | doi:10.1038/s41586-025-09746-w
  
  Abstract

  De novo enzyme design seeks to build proteins containing ideal active sites with catalytic residues surrounding and stabilizing the transition state(s) of the target chemical reaction. The generative artificial intelligence method RFdiffusion solves this problem, but requires specifying both the sequence position and backbone coordinates for each catalytic residue, limiting sampling. Here we introduce RFdiffusion2, which eliminates these requirements, and use it to design zinc metallohydrolases starting from quantum chemistry-derived active site geometries. From an initial set of 96 designs tested experimentally, the most active has a catalytic efficiency (k/K) of 16,000 M s, orders of magnitude higher than previously designed metallohydrolases. A second round of 96 designs yielded 3 additional highly active enzymes, with k/K values of up to 53,000 M s and a catalytic rate constant (k) of up to 1.5 s. The design models of the four most active designs differ from known structures and from each other, and the crystal structure of the most active design is very close to the design model, demonstrating the accuracy of the design method. The most active enzymes are predicted by PLACER and Chai-1 (ref. ) to have preorganized active sites that effectively position the substrate for nucleophilic attack by a water molecule activated by the bound metal. The ability to generate highly active enzymes directly from the computer, without experimental optimization, should enable a new generation of potent designer catalysts.

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• Computational design of serine hydrolases
  Enzymes
  Lauko A, Pellock SJ, Sumida KH, Anishchenko I, Juergens D, Ahern W, Jeung J, Shida AF, Hunt A, Kalvet I, Norn C, Humphreys IR, Jamieson C, Krishna R, Kipnis Y, Kang A, Brackenbrough E, Bera AK, Sankaran B, Houk KN, Baker D.
  Science, 2025 | doi:10.1126/science.adu2454
  
  Abstract

  The design of enzymes with complex active sites that mediate multistep reactions remains an outstanding challenge. With serine hydrolases as a model system, we combined the generative capabilities of RFdiffusion with an ensemble generation method for assessing active site preorganization at each step in the reaction to design enzymes starting from minimal active site descriptions. Experimental characterization revealed catalytic efficiencies (/) up to 2.2 × 10 M s and crystal structures that closely match the design models (Cα root mean square deviations <1 angstrom). Selection for structural compatibility across the reaction coordinate enabled identification of new catalysts remove with five different folds distinct from those of natural serine hydrolases. Our de novo approach provides insight into the geometric basis of catalysis and a roadmap for designing enzymes that catalyze multistep transformations.

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Collaborator-Led

• De novo design and evolution of an artificial metathase for cytoplasmic olefin metathesis
  Enzymes Enzymes
  Zou Z, Kalvet I, Lozhkin B, Morris E, Zhang K, Chen D, Ernst ML, Zhang X, Baker D, Ward TR.
  Nat Catal, 2025 | doi:10.1038/s41929-025-01436-0
  
  Abstract

  Artificial metalloenzymes present a promising avenue for abiotic catalysis within living systems. However, their in vivo application is currently limited by critical challenges, particularly in selecting suitable protein scaffolds capable of binding abiotic cofactors and maintaining catalytic activity in complex media. Here we address these limitations by introducing an artificial metathase-an artificial metalloenzyme designed for ring-closing metathesis-for whole-cell biocatalysis. Our approach integrates a tailored metal cofactor into a hyper-stable, de novo-designed protein. By combining computational design with genetic optimization, a binding affinity (  ≤ 0.2 μM) between the protein scaffold and cofactor is achieved through supramolecular anchoring. Directed evolution of the artificial metathase yielded variants exhibiting excellent catalytic performance (turnover number ≥1,000) and biocompatibility. This work represents a pronounced leap in the de novo design and in cellulo engineering of artificial metalloenzymes, paving the way for abiological catalysis in living systems.

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• De novo design of proteins housing excitonically coupled chlorophyll special pairs
  Enzymes
  Ennist NM, Wang S, Kennedy MA, Curti M, Sutherland GA, Vasilev C, Redler RL, Maffeis V, Shareef S, Sica AV, Hua AS, Deshmukh AP, Moyer AP, Hicks DR, Swartz AZ, Cacho RA, Novy N, Bera AK, Kang A, Sankaran B, Johnson MP, Phadkule A, Reppert M, Ekiert D, Bhabha G, Stewart L, Caram JR, Stoddard BL, Romero E, Hunter CN, Baker D.
  Nat Chem Biol, 2024 | doi:10.1038/s41589-024-01626-0
  
  Abstract

  Natural photosystems couple light harvesting to charge separation using a ‘special pair’ of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

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• Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site
  Enzymes
  Kalvet I, Ortmayer M, Zhao J, Crawshaw R, Ennist NM, Levy C, Roy A, Green AP, Baker D.
  J Am Chem Soc, 2023 | doi:10.1021/jacs.3c02742
  
  Abstract

  The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

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• Multimerization of an Alcohol Dehydrogenase by Fusion to a Designed Self-Assembling Protein Results in Enhanced Bioelectrocatalytic Operational Stability
  Enzymes Nanoparticles
  Bulutoglu B, Macazo FC, Bale J, King N, Baker D, Minteer SD, Banta S.
  ACS Appl Mater Interfaces, 2019 | doi:10.1021/acsami.9b04256
  
  Abstract

  Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.

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