Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening

Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this system particularly challenging to optimize. Yet, experimental screening of a small number of active-site variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers ( 2 104 M 1 s 1 and 102 s 1) for this anthropogenic reaction that compare favorably to those of modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within 2 kcal mol 1 and indicate that the enhanced activity is linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stabilityguidance of FuncLib by computational predictions of catalytic activity, as a generalized approach for computational enzyme designKnut and Alice Wallenberg Foundation (Wallenberg Academy Fellowship) 2018.0140Human Frontier Science Program RGP0041/2017FEDER Funds/Spanish Ministry of Science, Innovation and Universities BIO2015-66426-R RTI2018-097142-B-100FEDER/Junta de Andalucia - Consejeria de Economia y Conocimiento E.FQM.113.UGR18Swedish National Infrastructure for computing (SNAC) 2018/2-3 2019/2-

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.R. and B.S.), Spanish Ministry of Science and Innovation FEDER Funds Grants RTI2018-097142-B-100 and PID2021-124534OB-100 (J.M.S.R.), National Aeronautics and Space Administration (NASA) Grant 80NSSC18K1277 (B.S.) Grant RYC2021-031155-I (E.M.C.) from Spanish Ministry of Science and Innovation and NextGenerationEU/PRTR and Grant E-BIO-464-UGR-20 (E.M.C.) from FEDER Funds and Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucia. E.A.L. was a recipient of a postdoctoral fellowship from the regional Andalusian Government (2020_DOC_00541). We thank the “Centro de Supercomputacion” (ALHAMBRA-CSIRC) of the University of Granada for providing computational resources and Dr Valeria A. Risso for useful discussions, comments on the manuscript and help with the evolutionary and structural analyses of the rescuing mutations.Peer reviewe

Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral β-lactamase with a new active site that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M−1 s−1, kcat~635 s−1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed

Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral β-lactamase with a new active site that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M−1 s−1, kcat~635 s−1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed

Protection of Catalytic Cofactors by Polypeptides as a Driver for the Emergence of Primordial Enzymes

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.This work was supported by Human Frontier Science Program grant RGP0041/2017 (J.M.S.R. and E.A.G.), National Science Foundation grant 2032315 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), National Institutes of Health grant R01AR069137 (E.A.G.), Spanish Ministry of Science and Innovation/ FEDER Funds grant PID2021-124534OB-100 (J.M.S.R.), and grant PID2020-116261GB-I00 (J.A.G.)

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </div

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on E. coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted

Heme-binding enables allosteric modulationin an ancient TIM-barrel glycosidase

[EN] Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.his work was supported by Human Frontier Science Program Grant RGP0041 (J.M.S.-R., E.A.G., B.S., and S.C.L.K.), NIH grant R01AR069137 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), the Swedish Research Council (2019-03499) (S.C.L.K.), the Knut and Alice Wallenberg Foundation (2018.0140 and 2019.0431) (S.C.L.K.), Spanish Ministry of Economy and Competitiveness/FEDER Funds Grants BIO2015-66426-R (J.M.S.-R.) RTI2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.). The simulations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX partially funded by the Swedish Research Council through grant agreement no. 2016-07213. We acknowledge the Spanish Synchrotron Radiation Facility (ALBA, Barcelona) for the provision of synchrotron radiation facilities and the staff at XALOC beamline for their invaluable support. We are also grateful to Victoria Longobardo Polanco (Proteomic Unit, Institute of Parasitology and Biomedicine “López-Neyra”) for help with mass spectrometry experiments and data analyses and to Juan Román Luque Ortega (Molecular Interactions Facility, Centro de Investigaciones Biológicas Margarita Salas) for help with ultracentrifugation experiments and data analyses.Peer reviewe