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 beta-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 (k(cat)/K-M similar to 2.10(5) M-1 s(-1), k(cat)similar to 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.Human Frontier Science Program RGP0041/2017Spanish Government RTI-2018-097142-B100 EQC2019-006403-PFEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento E.FQM.113.UGR1

Consensus Design of an Evolved High-Redox Potential Laccase

Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.This study is based upon work funded by and the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016- 79106-R-Lignolution. BG-F was supported by a FPI national fellowship BES-2014-068887

J R SOC INTERFACE

Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biological function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-of-the-art computational and experimental approaches are revealing the crucial molecular details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temperatures and the possibility of additional phenotypic mutationsDepartment of Chemistry, BMC, Uppsala University, Box 576, 751 23 Uppsala, Sweden Departamento de Quimica Fisica, Facultad de Ciencias, University of Granada, 18071 Granada, SpainWallenberg Academy Fellowship to S.C.L.K. from the Knut and Alice Wallenberg Foundation (KAW 2013.0124)Grant RGP0041/2017 from the Human Frontier Science ProgramFEDER Funds and grant BIO2015–66426-R to J.M.S.R. from the Spanish Ministry of Economy and Competitivenes

X-ray evidence of a native state with increased compactness populated by tryptophan-less B. licheniformis β-lactamase

β-lactamases confer antibiotic resistance, one of the most serious world-wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class-A β-lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7-Å resolution X-ray structure, catalytic parameters, and thermodynamic stability of ESPΔW, an engineered mutant of ESP in which phenylalanine replaces the wild-type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis β-lactamases (RMSD = 0.4-1.2 Å). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESPΔW has a significantly smaller radius of gyration (Rg) than the other B. licheniformis β-lactamases characterized so far. Indeed, ESPΔW has the smallest Rg among 126 Class-A β-lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESPΔW proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class-A β-lactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold.Fil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Acierno, Juan Pablo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Capaldi, Stefano. Universita di Verona; ItaliaFil: Monaco, Hugo L.. Universita di Verona; ItaliaFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

ABSTRACT Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back 500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3- cyclopentanedione oxidation, a –diketone initiator commonly used in vinyl polymerization reactions. IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the -diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers

Hinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity

W.D.V.H. acknowledges support from National Institutes of Health (Grant: R01GM112077). S.B.O. acknowledges support from the Gordon and Betty Moore Foundations and National Science Foundation (Awards: 1715591 and 1901709). J.M.S.R. acknowledges support from Spanish Ministry of Economy and Competitiveness/FEDER Funds (Grants BIO2015-66426-R and RTI2018-097142-B-100) and the Human Frontier Science Program (Grant RGP0041/2017). V.A.R. acknowledges support from FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento (Grant E.FQM.113.UGR18). We would like to thank the beamline staff of ID30B of the ESRF (European Synchrotron Radiation Facility, Grenoble, France) for their assistance during data collection and the ESRF for the provision of time through proposals MX-2064.TEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.United States Department of Health & Human Services National Institutes of Health (NIH) - USA R01GM112077Gordon and Betty Moore FoundationsNational Science Foundation (NSF) 1715591 1901709Spanish Ministry of Economy and Competitiveness/FEDER Funds BIO2015-66426-R RTI2018-097142-B-100Human Frontier Science Program RGP0041/2017FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento E.FQM.113.UGR1

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 proteinmediated 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.Human Frontier Science Program grant RGP0041/2017National Science Foundation grant 2032315Department of Defense grant MURI W911NF-16-1-0372National Institutes of Health grant R01AR069137Spanish Ministry of Science and Innovation/ FEDER Funds grant PID2021-124534OB-100Grant PID2020-116261GB-I0

Evidence for a role of phenotypic mutations in virus adaptation

This work was supported by Spanish Ministry of Economy and Competitiveness/FEDER Funds Grant RTI2018-097142-B-100 and by Human Frontier Science Program Grant RGP0041/2017. Viral genome library preparation and Illumina sequencing were carried out at the IPBLN Genomics Facility (CSIC, Granada, Spain), and the assistance of Dr. Alicia Barroso del Jesus is gratefully acknowledged. We also thank Dr. Jon Beckwith and Dr. Dana Boyd (Harvard University) for kindly providing knockout strains used in this work.Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102257.Viruses interact extensively with the host molecular machinery, but the underlying mechanisms are poorly understood. Bacteriophage T7 recruits the small protein thioredoxin of the Escherichia coli host as an essential processivity factor for the viral DNA polymerase. We challenged the phage to propagate in a host in which thioredoxin had been extensively modified to hamper its recruitment. The virus adapted to the engineered host without losing the capability to propagate in the original host, but no genetic mutations were fixed in the thioredoxin binding domain of the viral DNA polymerase. Virus adaptation correlated with mutations in the viral RNA polymerase, supporting that promiscuous thioredoxin recruitmentwas enabled by phenotypicmutations caused by transcription errors. These results point to a mechanism of virus adaptation that may play a role in cross-species transmission.We propose that phenotypicmutations may generally contribute to the capability of viruses to evade antiviral strategiesSpanish Ministry of Economy and Competitiveness/FEDER Funds Grant RTI2018-097142-B-100Human Frontier Science Program Grant RGP0041/201

Correlation of Watch Antibiotic Consumption with a Gram-negative Bacteria Resistance: Analysis at a Country Level

Introduction: Bacterial antimicrobial resistance (AMR) to the antibiotics (ATB) has severe consequences for human health. The excess ATB consumption is one of the main causes of AMR. One of the World Health Organizations main objective in AMR control strategy is to limit the ATB irrational use, that is why, it proposed to classify the ATB in three groups: “Access”, “Watch”, and “Reserve”, being the latter two preserved for certain situations. The present work aims to know the level of ATB consumption and its correlation with the Gram-negative resistance in Argentina. Materials and Methods: Gram-negative bacteria resistance to “watch” the ATB groups (cephalosporins/carbapenem/fluoroquinolones) were explored for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Antibiotic consumption was expressed by the defined daily dose/1000 inhabitants for each ATB for one year period (2018). Resistance/consumption ratio was obtained by calculating the “R” for each region of the country, comparing them with the other countries. Results: E. coli resistance to 3rd generation cephalosporines was 8.68% and 16.51% in urine (U) and blood (B) samples, respectively, while to carbapenems was 0.08% (U) and 0.36% (B). Resistance of K. pneumoniae to 3 GC was 39.78% (U) and 52.45% (B) while to carbapenem was 9.03% (U) and 17.46% (B). P. aeruginosa resistance to fluoroquinolone and to carbapenems was 29.7% (U)/26.4% (B) and 17.7% (U)/19.9% (B), respectively. The resistance/consumption ratio was heterogenous within the country. Most of the populated areas patterns had similarities with the one observed in the less developed countries (mild-high resistance/mild-high consumption), while the ratio found in less densely populated areas, mimicked countries with the most rational use of ATB. Conclusion: In Argentina, Gram-negative bacteria showed overall high/mild resistance levels against the “Watch” ATB groups, with a largely variations among each region.Fil: Boni, Silvia. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Marin, Gustavo Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Campaña, Laura. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Marin, Lupe. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Marin, G.. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Risso Patron, Soledad. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Gabriel, Fernanda. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Corso, Alejandra. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Garay, Valeria. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; ArgentinaFil: Limeres, Manuel. Ministerio de Salud. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica; Argentin

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.This research was supported by FEDER Funds, grant BIO2015-66426-R from the Spanish Ministry of Economy and Competitiveness ( J.M.S.-R.), grant RGP0041/2017 from the Human Frontier Science Program ( J.M.S.-R. and E.A.G.) and National Institutes of Health 1R01AR069137 (E.A.G.), Department of Defence MURI W911NF-16-1-0372 (E.A.G.)