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

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.)

Engineering, evolution and folding of enzymes with natural and non natural activities

First, we have explored the relationship between in vitro and in vivo protein folding, in an evolutionary context. We have experimentally characterized the in vitro folding of a set of resurrected Precambrian and modern thioredoxins and found that, contrary to previous claims in the literature, the folding rates are not evolutionarily conserved. Thus, ancestral thioredoxins fold much faster in the test tube than their modern counterparts. Extensive mutational analyses have allowed us to identify mutation S74G as responsible for aggravating folding in E. coli thioredoxin. The evolutionary acceptance of this mutation is interpreted as an example of degradation of ancestral features at the molecular level. We propose that unassisted and efficient primordial folding was linked to fast folding encoded at the sequence/structure level. Once an efficient assistance machinery had emerged, mutations that impaired ancient sequence/structure determinants of folding efficiency could be accepted, since those determinants were no longer necessary. We conclude that in vitro and in vivo folding landscapes are disconnected and question the biologically relevance of the in vitro folding rate determinations, except as related to heterologous folding efficiency (see below). In the second part of this thesis, we have addressed a pivotal and common problem in biotechnology, the inefficient heterologous expression of proteins. As a model system thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiotic bacteria of flashlight fish, has been used. Our results demonstrate its slow in vitro folding (it takes several hours to reach the native state) and inefficient expression in E. coli, leading mostly to insoluble protein. By using a few back-to-the-ancestral mutations at positions selected by computational modelling of the unassisted folding landscape we were able to rescue its inefficient expression. Our results support that the folding of proteins in foreign hosts may be akin to some extent to unassisted folding due to the absence of coevolution of the recombinant protein with the natural chaperones of the new host. More generally, our results provide an approach based on sequence engineering to rescue inefficient heterologous expression with a minimal protein perturbation.En primer lugar, hemos explorado la relación entre el plegamiento de proteínas in vitro e in vivo, en un contexto evolutivo. Hemos caracterizado experimentalmente el plegamiento in vitro de un conjunto de tiorredoxinas precámbricas y modernas y hemos descubierto que, al contrario de lo que ha sido afirmado recientemente en la literatura, la velocidad de plegamiento no ha sido conservada a lo largo de la evolución. Las tiorredoxinas ancestrales se pliegan mucho más rápido in vitro que sus homólogas modernas. Un extenso análisis mutacional nos ha permitido identificar que la mutación S74G es la responsable de hacer más lento el plegamiento en la tiorredoxina de E. coli. La aceptación evolutiva de esta mutación se interpreta como un ejemplo de degradación de rasgos ancestrales a nivel molecular. En nuestro trabajo, se propone que el plegamiento primordial, supuestamente no asistido y eficiente, está ligado a un plegamiento rápido que debe estar codificado a nivel de secuencia / estructura. Pero, una vez surgió la maquinaria de asistencia para el plegamiento, se aceptaron mutaciones que dañaban los determinantes de secuencia / estructura que codificaban para un plegamiento rápido in vitro, ya que esta característica dejó de ser necesaria. Por tanto, en este trabajo se concluye que los paisajes de plegamiento in vitro e in vivo están desconectados y se cuestiona la importancia biológica de la velocidad de plegamiento in vitro, excepto en los casos de expresión heteróloga (ver más abajo). En la segunda parte de esta tesis, hemos abordado uno de los problemas más comunes en biotecnología, la ineficiente expresión heteróloga de proteínas. Como sistema modelo, se ha utilizado la tiorredoxina de Candidatus Photodesmus katoptron, una bacteria simbiótica no cultivable del pez linterna. Nuestros resultados demuestran que esta proteína presenta un plegamiento in vitro muy lento (necesita varias horas para alcanzar su estado nativo) y su expresión es ineficaz en E. coli, lo que lleva principalmente a la obtención de proteína insoluble. Mediante el uso de mutaciones de vuelta al ancestro, en determinadas posiciones seleccionadas por un modelo computacional del paisaje de plegamiento, pudimos rescatar su expresión. Nuestros resultados apoyan que el plegamiento de proteínas en un huésped diferente, puede ser similar, en cierta medida, al plegamiento no asistido debido a la ausencia de coevolución de la proteína recombinante con las chaperonas naturales del nuevo huésped. De manera más general, nuestros resultados desarrollan un enfoque basado en la ingeniería de secuencias para rescatar la expresión heteróloga ineficaz con una perturbación mínima de la proteína.Tesis Univ. Granada.FEDER Funds, grant BIO2015-66426-R from the Spanish Ministry of Economy and CompetitivenessGrant RGP0041/2017 from the Human Frontier Science ProgramUppsala Universit

Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression.

This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.-R. and E.A.G.), National Science Foundation Award #2032315 (E.A.G.), National Institutes of Health Award #R01AR069137 (E.A.G.), Department of Defense MURI Award #W911NF-16-1-0372 (E.A.G.), Spanish Ministry of Science and Innovation/FEDER Funds Grants RTI-2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.) and the Science, Engineering and Research Board (SERB, India) Grant MTR/2019/000392 (A.N.N.). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and the staff at ID23-1 beamline for assistance during data collection. J.M.S.R. designed the research. G.G.-A. purified the modern/ancestral chimeras and the thioredoxin variants; she also performed and analysed the experiments aimed at determining their folding kinetics and biomolecular properties. V.A.R. performed experiments addressed at determining the efficiency of heterologous expression and provided essential input for the molecular interpretation of mutational effects on expression efficiency. E.A.G. provided essential input for the evolutionary interpretation of the data. J.A.G. determined the X-ray structure of the symbiont protein and provided essential input regarding its interpretation and implications. A.N.N. performed the computational simulations of the folding landscape for thioredoxins and provided essential input regarding their engineering implications. B.I.M. and J.M.S.-R. directed the project. J.M.S.-R. wrote the first draft of the manuscript to which V.A.R. J.A.G. A.N.N. and B.I.M. added crucial paragraphs and sections. All authors discussed the manuscript, suggested modifications and improvements, and contributed to the final version. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1–2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations.National Science Foundation 2032315National Institutes of Health 01AR069137U.S. Department of Defense 911NF-16-1-0372Human Frontier Science Program RGP0041/2017European Synchrotron Radiation FacilityScience and Engineering Research Board MTR/2019/000392Ministerio de Ciencia e Innovación RTI-2018-097142-B-10

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-function al enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus con tributes 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 pro line. 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 seques tered 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 poten tial of mutation-induced leakage in metabolic engineering is notedHuman Frontier Science Program RGP0041/2017Spanish Government RTI2018-097142-B-100Ministry of Science and Innovation, Spain (MICINN) 80NSSC18K1277European CommissionJunta de AndaluciaRegional Andalusian Government E-BIO-464-UGR-20 2020_DOC_0054