Resurrected ancestral proteins as scaffolds for enzyme engineering and evolution

Abstract

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.