Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Pyramidalization of the Carboxamide sp2-Center in Peptide Structures.

A CSD search in the Cambridge Crystallographic Database for the substructure N-CαH-C'(═O)-N gave 24,180 peptide structures for analysis of the pyramidalization of the sp2-hybridized carboxamide group C'(═O)NCα, which had not been investigated before. The dependence of the pyramidalization θ = O-N-C'-Cα on the rotation angle ψ = O═C'-Cα-N about bond C'-Cα resulted in a curve with three maxima, three minima, and six zero-crossings. Surprisingly, the ψ/θ analysis of the individual amino acid building blocks showed that all of them exhibited similar curves, irrespective of their different R substituents. This unusual behavior is explained by a 3-fold short-range potential set up by the three covalent bonds, emanating from Cα. The tie-up of the rotation angle ψ and the pyramidalization θ in a rigid coupling is remarkable. In the 24,180 peptide structures, subjected to X-ray crystallography, there is no dynamics. For peptides in solution, the rotation/pyramidalization curve ψ/θav determines the degree of pyramidalization θ, when the rotation angle ψ runs through a full 360° circle. Density functional theory (DFT) calculations of alaninamide supported the analysis.

Synthesis, crystal structure and in-silico evaluation of arylsulfonamide Schiff bases for potential activity against colon cancer.

This report presents a comprehensive investigation into the synthesis and characterization of Schiff base compounds derived from benzenesulfonamide. The synthesis process, involved the reaction between N-cycloamino-2-sulfanilamide and various substituted o-salicylaldehydes, resulted in a set of compounds that were subjected to rigorous characterization using advanced spectral techniques, including 1H NMR, 13C NMR and FT-IR spectroscopy, and single-crystal X-ray diffraction. Furthermore, an in-depth assessment of the synthesized compounds was conducted through Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) analysis, in conjunction with docking studies, to elucidate their pharmacokinetic profiles and potential. Impressively, the ADMET analysis showcased encouraging drug-likeness properties of the newly synthesized Schiff bases. These computational findings were substantiated by molecular properties derived from density functional theory (DFT) calculations using the B3LYP/6-31G* method within the Jaguar Module of Schrödinger 2023-2 from Maestro (Schrodinger LLC, New York, USA). The exploration of frontier molecular orbitals (HOMO and LUMO) enabled the computation of global reactivity descriptors (GRDs), encompassing charge separation (Egap) and global softness (S). Notably, within this analysis, one Schiff base, namely, 4-bromo-2-{N-[2-(pyrrolidine-1-sulfonyl)phenyl]carboximidoyl}phenol, 20, emerged with the smallest charge separation (ΔEgap = 3.5780 eV), signifying heightened potential for biological properties. Conversely, 4-bromo-2-{N-[2-(piperidine-1-sulfonyl)phenyl]carboximidoyl}phenol, 17, exhibited the largest charge separation (ΔEgap = 4.9242 eV), implying a relatively lower propensity for biological activity. Moreover, the synthesized Schiff bases displayed remarkeable inhibition of tankyrase poly(ADP-ribose) polymerase enzymes, integral in colon cancer, surpassing the efficacy of a standard drug used for the same purpose. Additionally, their bioavailability scores aligned closely with established medications such as trifluridine and 5-fluorouracil. The exploration of molecular electrostatic potential through colour mapping delved into the electronic behaviour and reactivity tendencies intrinsic to this diverse range of molecules.

Approach to the "Missing" Diarylsilylene: Formation, Characterization, and Intramolecular C-H Bond Activation of Blue Diarylsilylenes with Bulky Rind Groups.

The treatment of the bulky Rind-based dibromosilanes, (Rind)2SiBr2 (2) [Rind = 1,1,7,7-tetra-R1-3,3,5,5-tetra-R2-s-hydrindacen-4-yl: EMind (a: R1 = Et, R2 = Me) and Eind (b: R1 = R2 = Et)], with two equivalents of tBuLi in Et2O at low temperatures resulted in the formation of blue solutions derived from the diarylsilylenes, (Rind)2Si: (3). Upon warming the solutions above -20 °C, the blue color gradually faded, accompanying the decomposition of 3 and yielding cyclic hydrosilanes (4) via intramolecular C-H bond insertion at the Si(II) center. The molecular structures of the bulky Eind-based 3b and 4b were confirmed by X-ray crystallography. Thus, at -20 °C, blue crystals were formed (Crystal-A), which were identified as mixed crystals of 3b and 4b. Additionally, colorless crystals of 4b as a singular component were isolated (Crystal-B), whose structure was also determined by an X-ray diffraction analysis. Although the isolation of 3 was difficult due to their thermally labile nature, their structural characteristics and electronic properties were discussed based on the experimental findings complemented by computational results. We also examined the hydrolysis of 3b to afford the silanol, (Eind)2SiH(OH) (5b).

The influence of model building schemes and molecular dynamics sampling on QM-cluster models: the chorismate mutase case study.

Most QM-cluster models of enzymes are constructed based on X-ray crystal structures, which limits comparison to in vivo structure and mechanism. The active site of chorismate mutase from Bacillus subtilis and the enzymatic transformation of chorismate to prephenate is used as a case study to guide construction of QM-cluster models built first from the X-ray crystal structure, then from molecular dynamics (MD) simulation snapshots. The Residue Interaction Network ResidUe Selector (RINRUS) software toolkit, developed by our group to simplify and automate the construction of QM-cluster models, is expanded to handle MD to QM-cluster model workflows. Several options, some employing novel topological clustering from residue interaction network (RIN) information, are evaluated for generating conformational clustering from MD simulation. RINRUS then generates a statistical thermodynamic framework for QM-cluster modeling of the chorismate mutase mechanism via refining 250 MD frames with density functional theory (DFT). The 250 QM-cluster models sampled provide a mean ΔG‡ of 10.3 ± 2.6 kcal mol-1 compared to the experimental value of 15.4 kcal mol-1 at 25 °C. While the difference between theory and experiment is consequential, the level of theory used is modest and therefore "chemical" accuracy is unexpected. More important are the comparisons made between QM-cluster models designed from the X-ray crystal structure versus those from MD frames. The large variations in kinetic and thermodynamic properties arise from geometric changes in the ensemble of QM-cluster models, rather from the composition of the QM-cluster models or from the active site-solvent interface. The findings open the way for further quantitative and reproducible calibration in the field of computational enzymology using the model construction framework afforded with the RINRUS software toolkit.

Toward More Selective Antibiotic Inhibitors: A Structural View of the Complexed Binding Pocket of E. coli Peptide Deformylase.

Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.

Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, 27 inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.

PURA syndrome-causing mutations impair PUR-domain integrity and affect P-body association.

Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

PURA syndrome is a neurodevelopmental disorder that affects about 650 patients worldwide, resulting in a range of symptoms including neurodevelopmental delays, intellectual disability, muscle weakness, seizures, and eating difficulties. The condition is caused by a mutated gene that codes for a protein called PURA. PURA binds RNA ­ the molecule that carries genetic information so it can be translated into proteins ­ and has roles in regulating the production of new proteins. Contrary to other conditions that result from mutations in a single gene, PURA syndrome patients show 'high penetrance', meaning almost every reported mutation in the gene leads to symptoms. Proske, Janowski et al. wanted to understand the molecular basis for this high penetrance. To find out more, the researchers first examined how patient mutations affected the location of the PURA in the cell, using human cells grown in the laboratory. Normally, PURA travels to P-bodies, which are groupings of RNA and proteins involved in regulating which genes get translated into proteins. The researchers found that in cells carrying PURA syndrome mutations, PURA failed to move adequately to P-bodies. To find out how this 'mislocalization' might happen, Proske, Janowski et al. tested how different mutations affected the three-dimensional folding of PURA. These analyses showed that the mutations impair the protein's folding and thereby disrupt PURA's ability to bind RNA, which may explain why mutant PURA cannot localize correctly. Proske, Janowski et al. describe the molecular abnormalities of PURA underlying this disorder and show how molecular analysis of patient mutations can reveal the mechanisms of a disease at the cell level. The results show that the impact of mutations on the structural integrity of the protein, which affects its ability to bind RNA, are likely key to the symptoms of the syndrome. Additionally, their approach used establishes a way to predict and test mutations that will cause PURA syndrome. This may help to develop diagnostic tools for this condition.

Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to ß-glucans.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to ß-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Synthetic access to diverse thiazetidines via a one-pot microwave assisted telescopic approach and their interaction with biomolecules.

A one-pot microwave assisted telescopic approach is reported for the chemo-selective synthesis of substituted 1,3-thiazetidines using readily available 2-aminopyridines/pyrazines/pyrimidine, substituted isothiocyanates and 1,2-dihalomethanes. The procedure involves thiourea formation from 2-aminopyridines/pyrazines/pyrimidine with the substituted isothiocyanates followed by a base catalysed nucleophilic attack of the CS bond on the 1,2-dihalomethane. Subsequently, a cyclization reaction occurs to yield substituted 1,3-thiazetidines. These four membered strained ring systems are reported to possess broad substrate scope with high functional group tolerance. The above synthetic sequence for the formation of four membered heterocycles is proven to be a modular and straightforward approach. Further the mechanistic pathway for the formation of 1,3-thiazetidines was supported by computational evaluations and X-ray crystallography analyses. The relevance of these thiazetidines in biological applications is evaluated by studying their ability to bind bio-macromolecules like proteins and nucleic acids.

Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate and harbors a Zn2+ ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives. High-resolution crystal structures of GmhA complexed to two N-formyl hydroxamate inhibitors confirmed the binding interactions to a central Zn2+ ion coordination site. Some of these compounds were found to be nanomolar inhibitors of GmhA. While devoid of HepG2 cytotoxicity and antibacterial activity of their own, they demonstrated in vitro lipopolysaccharide heptosylation inhibition in Enterobacteriaceae as well as the potentiation of erythromycin and rifampicin in a wild-type Escherichia coli strain. These inhibitors pave the way for a novel treatment of Gram-negative infections.

Influence of pozzolanic addition on strength and microstructure of metakaolin-based concrete.

The intent of this study is to explore the physical properties and long-term performance of concrete made with metakaolin (MK) as a binder, using microsilica (MS) and nanosilica (NS) as substitutes for a portion of the ordinary Portland cement (OPC) content. The dosage of MS was varied from 5% to 15% for OPC-MK-MS blends, and the dosage of NS was varied from 0.5% to 1.5% for OPC-MK-NS blends. Incorporation of these pozzolans accelerated the hardening process and reduced the flowability, consistency, and setting time of the cement paste. In addition, it produced a denser matrix, improving the strength of the concrete matrix, as confirmed by scanning electron microscopy and X-ray diffraction analysis. The use of MS enhanced the strength by 10.37%, and the utilization of NS increased the strength by 11.48% at 28 days. It also reduced the penetrability of the matrix with a maximum reduction in the water absorption (35.82%) and improved the resistance to the sulfate attack for specimens containing 1% NS in the presence of 10% MK. Based on these results, NS in the presence of MK can be used to obtain cementitious structures with the enhanced strength and durability.

New pimarane diterpenoids with antibacterial activity from fungus Arthrinium sp. ZS03.

A comprehensive chemical study of the endophytic fungus Arthrinium sp. ZS03, associated with Acorus tatarinowii Schott, yielded eleven pimarane diterpenoids (compounds 1-11), including seven novel compounds designated arthrinoids A-G (1-7). The determination of their structures and absolute configurations was achieved through extensive spectroscopic techniques, quantum chemical calculations of electronic circular dichroism (ECD), and single-crystal X-ray diffraction analysis. Furthermore, 7 demonstrated inhibitory activity against Klebsiella pneumoniae, comparable to the reference antibiotic amikacin, with a minimum inhibitory concentration (MIC) of 8 µg·mL-1.

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source.

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing the catalytic efficiency of de novo enzymes by several orders of magnitude without relying on directed evolution and high-throughput screening. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (kcat/KM 125 M-1 s-1) and KE70 (kcat/KM 57 M-1 s-1), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display kcat/KM values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models, with catalytic contacts present as designed and transition-state root-mean-square deviations of ≤0.65 Å. Our work shows how ensemble-based design can generate efficient artificial enzymes by exploiting the true conformational ensemble to design improved active sites.