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1. Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

   https://doi.org/10.1038/s41467-020-18619-x  

   Broom, Aron; Rakotoharisoa, Rojo V; Thompson, Michael C; Zarifi, Niayesh; Nguyen, Erin; Mukhametzhanov, Nurzhan; Liu, Lin; Fraser, James S; Chica, Roberto A (December 2020, Nature Communications)

   Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k_cat/K_M146 M⁻¹s⁻¹). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k_cat/K_M103,000 M⁻¹s⁻¹) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. 

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2. Investigating the cis- regulatory basis of C ₃ and C ₄ photosynthesis in grasses at single-cell resolution

   https://doi.org/10.1073/pnas.2402781121  

   Mendieta, John Pablo; Tu, Xiaoyu; Jiang, Daiquan; Yan, Haidong; Zhang, Xuan; Marand, Alexandre P; Zhong, Silin; Schmitz, Robert J (October 2024, Proceedings of the National Academy of Sciences)

   While considerable knowledge exists about the enzymes pivotal for C₄photosynthesis, much less is known about thecis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C₄enzymes for five different grass species. This study spans four C₄species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-dependent malic enzyme),Panicum miliaceum(NAD-dependent malic enzyme),Urochloa fusca(phosphoenolpyruvate carboxykinase), along with the C₃outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C₄species and those unique to C₄subtypes, measuring cell-type-specific biases for C₄enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C₄evolution. Besides promoter proximal ACRs, we found that, on average, C₄genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C₄evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution ofcis-regulation at these C₄loci. This study illuminates the dynamic and complex nature ofcis-regulatory elements evolution in C₄photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C₃crop performance under changing climatic conditions. 

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3. 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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4. Solar‐Driven CO ₂ Conversion via Optimized Photothermal Catalysis in a Lotus Pod Structure

   https://doi.org/10.1002/anie.202305251  

   Wang, Hongmin; Fu, Shuting; Shang, Bo; Jeon, Sungho; Zhong, Yiren; Harmon, Nia J.; Choi, Chungseok; Stach, Eric A.; Wang, Hailiang (July 2023, Angewandte Chemie International Edition)

   Abstract Photothermal CO₂reduction is one of the most promising routes to efficiently utilize solar energy for fuel production at high rates. However, this reaction is currently limited by underdeveloped catalysts with low photothermal conversion efficiency, insufficient exposure of active sites, low active material loading, and high material cost. Herein, we report a potassium‐modified carbon‐supported cobalt (K⁺−Co−C) catalyst mimicking the structure of a lotus pod that addresses these challenges. As a result of the designed lotus‐pod structure which features an efficient photothermal C substrate with hierarchical pores, an intimate Co/C interface with covalent bonding, and exposed Co catalytic sites with optimized CO binding strength, the K⁺−Co−C catalyst shows a record‐high photothermal CO₂hydrogenation rate of 758 mmol g_cat⁻¹ h⁻¹(2871 mmol g_Co⁻¹ h⁻¹) with a 99.8 % selectivity for CO, three orders of magnitude higher than typical photochemical CO₂reduction reactions. We further demonstrate with this catalyst effective CO₂conversion under natural sunlight one hour before sunset during the winter season, putting forward an important step towards practical solar fuel production. 

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5. Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

   https://doi.org/10.1073/pnas.1901864116  

   Dasgupta, Medhanjali; Budday, Dominik; de_Oliveira, Saulo_H P; Madzelan, Peter; Marchany-Rivera, Darya; Seravalli, Javier; Hayes, Brandon; Sierra, Raymond G; Boutet, Sébastien; Hunter, Mark S; et al (December 2019, Proceedings of the National Academy of Sciences)

   How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis. 

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