Resurrected ancestral proteins as scaffolds for enzyme engineering and evolution

Enzymes are extraordinary efficient natural molecular machines that catalyze chemical reactions and transformations that sustain life in all organisms. Decades of intensive research have led to significant advances in the study of enzymes. Researchers have developed sophisticated methodologies and approaches to extensively study and gain an in-depth understanding of the molecular basis of enzyme structure, dynamics, function, and regulation. As a result, it is now possible to accurately describe the physiochemical implications of every element involved in almost every enzyme’s active site during the specific molecular processes that drive the catalytic reactions. Moreover, the extensive knowledge about enzymatic catalysis has allowed us to understand how enzymes have evolved during billions of years of natural selection to catalyze chemical reactions with proficient efficiency, specificity and selectivity towards the chemical transformations and their substrates. Overall, the study of enzymes has provided a fascinating window into the molecular machinery of life. But also, it has allowed researchers to accumulate a solid scientific knowledge base that enables to engineer and tune the molecular architecture of enzymes towards designing efficient artificial and modified versions of tailored enzymes for catalyzing chemical reactions of biotechnological and biomedical interest. However, despite our deep knowledge and advanced understanding about the fundamentals and evolution of enzymatic catalysis, one elemental question remains unanswered – How enzymatic catalysis firstly emerged and evolved at the origin of proteins and enzymes? Understanding the molecular mechanisms underlying the evolutionary emergence of new enzymatic catalysis would not only be essential to understand the birth of enzymes and its implications in the origins of life. But also, it would be critical to design new biotechnological approaches inspired in these molecular mechanisms to efficiently design and generate novel enzymes to catalyze artificial unnatural chemical reactions of interest. Yet, the study of modern enzymes with the aim to shed some light on this fundamental question has not provided significant advances. In this thesis, we propose the hypothesis that resurrected ancestral proteins might be better scaffolds than their modern counterparts to study and understand the emergence of enzymatic catalysis. Ancestral active sites and their molecular architectures would be more useful to reveal and study the minimal requirements for catalysis. But also, ancestral proteins might be better starting points for engineering novel active sites to catalyze artificial unnatural chemical reactions. Advances in both directions may help us to reveal the molecular processes that drive the emergence of new catalysis in nature. Ancestral proteins then show the potential to have a profound impact in our understanding about enzyme catalysis, with critical implications in our knowledge about the origins of life and our capacity to develop new artificial enzymes. In order to validate our hypothesis, we have performed several experiments with different resurrected ancestral protein systems aiming to evolve a de novo artificial active site, as well as to understand how primordial levels of cofactor-dependent catalysis are promoted in an unevolved ancestral molecular scaffold. In the first part of the thesis, we describe the evolution of an artificial de novo active site, previously engineered in a resurrected ancestral β-lactamase scaffold, by means of computational and experimental low-throughput screenings. As a result, we have demonstrated how mutations in residues directly involved in a de novo active site or how the introduction of new additional residues in the protein sequence may improve the geometrical preorganization of the active site and generate new interactions that enhance the stabilization of the reaction transition state and promote low initial levels of activity to reach an efficient enzymatic catalysis comparable to natural enzymes. These results have direct implications in protein engineering and de novo enzyme design. But also, it provides new insights about the evolutionary processes that may led the early optimization of novel active sites during the emergence of enzymatic catalysis. In the second part of the thesis, we have resurrected an ancestral glycosidase protein with a typical TIM-barrel fold that displays unusual biochemical and biophysical features. Mainly, our ancestral TIM-barrel shows the ability to bind a molecule of the redox cofactor heme in a highly flexible region of the barrel architecture. Upon heme binding, the ancestral TIM-barrel displays a general rigidification of its structure, an allosteric modulation of its natural enzymatic activity and an unnatural novel peroxidase activity based on the redox catalytic power of heme. As a result, the ancestral heme binding TIM-barrel protein demonstrates the potential of resurrected proteins as scaffolds to harbor unusual combinations of properties of evolutionary and biotechnological interest. Additionally, the study of our redox active TIM-barrel provides new insights about the role cofactor protection in the emergence of proteins and enzymatic catalysis during the origin of life. Overall, the results presented in this thesis support the hypothesis that resurrected ancestral proteins may serve as superior scaffolds for enzyme engineering and evolutionary studies, aimed to better understand the emergence of enzymatic catalysis during the origin of life.Las enzimas son máquinas moleculares naturales extraordinariamente eficientes que catalizan las reacciones y transformaciones químicas que sustentan la vida de todos los organismos. Décadas de investigación intensiva han logrado avances significativos en el estudio de las enzimas. Los investigadores han desarrollado metodologías y aproximaciones sofisticadas para estudiar de manera extensiva y conseguir un conocimiento en profundidad sobre sobre las bases moleculares de la estructura, dinámica, función y regulación de las enzimas. Como resultado, hoy en día es posible describir de forma precisa las implicaciones fisicoquímicas de cada elemento involucrado en el sitio activo de prácticamente cualquier enzima durante los procesos moleculares específicos que permiten las reacciones catalíticas. Además, el conocimiento extensivo sobre la catálisis enzimática nos ha permitido entender cómo las enzimas han evolucionado durante miles de millones de años de selección natural para catalizar reacciones químicas con eficiencia, especificidad y selectividad excelentes con respecto a las reacciones de transformación y sus sustratos. En general, el estudio de las enzimas ha abierto una fascinante ventana a la maquinaria molecular de la vida. Pero, además, ha permitido a los investigadores acumular una sólida base de conocimiento científico que podemos aplicar en el diseño, modificación y optimización de la arquitectura molecular de las enzimas con el objetivo de diseñar versiones artificiales y modificadas de enzimas a medida para catalizar reacciones químicas de interés biotecnológico y biomédico. A pesar del extenso y avanzado conocimiento sobre los fundamentos y la evolución de la catálisis enzimática, sigue habiendo una pregunta elemental sin respuesta con respecto al estudio de las enzimas: ¿Cómo emergió y evolucionó la catálisis enzimática por primera vez durante el origen de las proteínas y las enzimas? La capacidad de entender los mecanismos moleculares que subyacen a la emergencia evolutiva de nuevas capacidades catalíticas en enzimas no solo es fundamental para entender el nacimiento de las enzimas y sus implicaciones en el origen de la vida. Además, es crítica para diseñar nuevas aproximaciones biotecnológicas inspiradas en estos mecanismos moleculares con el objetivo de diseñar y generar de forma eficiente nuevas enzimas para catalizar reacciones químicas artificiales no naturales de interés. Sin embargo, el estudio basado en enzimas modernas, encontradas en los organismos actuales, con el objetivo de responder a esta pregunta fundamental no logrado avances significativos. En esta tesis proponemos la hipótesis de que las proteínas ancestrales resucitadas podrían funcionar como mejores “andamios moleculares”, en comparación con sus homologas modernas, para estudiar y comprender la emergencia de la catálisis enzimática. El estudio de sitios activos ancestrales y sus arquitecturas moleculares podría ser más útil para revelar y estudiar los requerimientos mínimos necesarios para la catálisis enzimática. Además, las proteínas ancestrales podrían ser mejores puntos de inicio para el diseño de sitios nuevos sitios activos para catalizar reacciones químicas no naturales artificiales. En este sentido, lograr avances en ambas direcciones tendría importantes implicaciones para entender y revelar los procesos moleculares que dirigen la emergencia de nuevas catálisis enzimáticas en la naturaleza. Por lo tanto, las proteínas ancestrales muestran de potencial de tener un profundo impacto en nuestra comprensión sobre la catálisis enzimática, con implicaciones críticas en nuestro conocimiento sobre el origen de la vida y la capacidad de desarrollar nuevas enzimas artificiales. Para validar nuestra hipótesis, hemos realizado diferentes experimentos con diferentes sistemas de proteínas ancestrales resucitadas con el objetivo de evolucionar un sitio activo artificial de novo y de entender cómo niveles primordiales de catálisis dependiente de un cofactor son mejorados en un andamio molecular ancestral sin evolucionar. En la primera parte de la tesis describimos la evolución de un sitio activo artificial de novo, previamente diseñado en el andamio molecular de una β-lactamasa ancestral mediante cribados computacionales y experimentales de bajo número. Como resultado, hemos demostrado cómo mutaciones en residuos directamente involucrados en el sitio activo artificial o cómo la introducción de nuevos residuos adicionales en la secuencia de la proteína puede mejorar la preorganización geométrica del sitio activo y generar nuevas interacciones que aumentan la estabilización del estado de transición y mejoran los bajos niveles de actividad enzimática para llegar a una catálisis enzimática eficiente comparable a la de enzimas naturales. Estos resultados tienen implicaciones inmediatas en ingeniería de proteínas y el diseño de novo de enzimas. Pero, adicionalmente, aporta nuevo conocimiento sobre los mecanismos evolutivos que pudieron dar lugar a la optimización temprana de sitios activos nuevos durante la emergencia de la catálisis enzimática. En la segunda parte de esta tesis, hemos resucitado una glicosidasa ancestral que presenta un plegamiento típico en forma de barril TIM y que muestra unas propiedades bioquímicas y biofísicas inusuales. Principalmente, nuestro barril TIM ancestral muestra la capacidad de unir una molécula del cofactor redox hemo en una región excepcionalmente flexible de arquitectura del barril. La unión del hemo da lugar a un aumento general de la rigidez de la estructura de la proteína, a una modulación alostérica de la actividad natural de la enzima y a la generación de una actividad peroxidasa nueva no natural basada en el poder catalítico redox intrínseco del hemo. Como resultado, nuestra proteína ancestral con estructura de barril TIM y con la capacidad de unir hemo demuestra el potencial de la resurrección ancestral de proteínas como andamios que muestran combinaciones inusuales de propiedades con interés biotecnológico. Adicionalmente, el estudio de nuestro barril TIM con actividad redox aporta nuevos puntos de vista sobre el papel de la protección de los cofactores durante la emergencia de las proteínas y la catálisis enzimática durante en origen de la vida. En general, los resultados presentados en esta tesis apoyan la hipótesis de que las proteínas ancestrales resucitadas pueden servir como mejores andamiajes moleculares en la ingeniería y estudios evolutivos de enzimas, dirigidos a lograr un mayor conocimiento sobre la emergencia de la catálisis enzimática durante el origen de la vida.Tesis Univ. Granada

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

Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems

We thank all members of the NTG laboratory for helpful discussions during the development of this project. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Supplementary Data are available at NAR Online: https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkaa1149#supplementary-dataBacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnological applications.Spanish Ministerio de Ciencia, Innovacion y UniversidadesEuropean Commission BIO2017-82244-PFPU predoctoral fellowship grant from the Ministerio de Economia y Competitividad FPU15/02714FPU predoctoral fellowship grant from the Ministerio de Ciencia, Innovacion y Universidades FPU17/0508

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

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