The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

DIRECTED EVOLUTION FOR THE DESIGN OF NEW CATALYSTS

Introducing new catalytic function into existing proteins provide ways of understanding the fundamental principles of enzyme catalysis and has gained recognition as a leading tool for understanding protein folding, structure, and function. De novo computational design coupled with directed evolution can yield catalysts with novel functions and improved catalytic rate enhancement of enzymes.1-2 Minimalist protein design that focuses on the bare minimum requirements to achieve activity presents several important advantages. By utilizing basic physico-chemical properties and strategic placing of only few highly active residues one can feasibly sample a very large variety of possible catalysts. In more general terms minimalist approach looks for the mere possibility of catalysis, rather than trying to identify the most active catalyst possible. Even very basic designs that utilize a single residue introduced into non-enzymatic proteins or peptide bundles are surprisingly active. No complex calculations need to be set up and even a beginner can master this technique in a very short time. An enzyme nicknamed AlleyCatE is an allosterically regulated catalyst of ester hydrolysis that was generated by introducing a single histidine residue into a non-enzymatic protein calmodulin (CaM). The catalytic efficiency of the resulting enzyme is higher than that of any other rationally designed p-nitrophenylesterase and is on par with some catalytic antibodies

Computational strategies for the design of new enzymatic functions

In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels–Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.This work was supported by the Spanish Ministerio de Economía y Competitividad for project CTQ2012-36253-C03, Universitat Jaume I – Spain (project P1·1B2014-26), Generalitat Valenciana – Spain (PROMETEOII/2014/022 and ACOMP/2014/277 projects), Polish National Center for Science (NCN) (grant 2011/02/A/ST4/00246, 2012−2017), the Polish Ministry of Science and Higher Education (“Iuventus Plus” program project no. 0478/IP3/2015/73, 2015-2016) and the USA National Institute of Health (ref. NIH R01 GM065368). Authors acknowledge computational resources from the Servei d’Informàtica of Universitat de València on the ‘Tirant’ supercomputer and the Servei d’Informat̀ica of Universitat Jaume I

Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole

Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole

Coordination Geometries in Metallobundles Enforcing Oxidative and Hydrolytic Catalysis and Designing Biomaterials for Use As Antimicrobials

De novo protein design permits discovery of intricate folds and functions emulating natural enzymes in simpler, yet robust model constructs. Helical bundles serve as premier scaffolds to incorporate diverse reactivities from oxidative, reductive, to hydrolytic transformations observed in much grander O2-utilizing metalloproteins such as radical-generating ribonucleotide reductases and catalases. One notable family of de novo proteins is the Due Ferri or DF series of four-helix bundles providing a dinuclear site for metal incorporation and are amenable for tailoring active site reactivity. As reactive oxygen species and radical-based intermediates are prevalent and necessary for steering life-essential processes through reactive radicals stabilized by metals, we were curious to understand how the nature and number of metal ions influence sequestration of such species. We investigated the influence metal has on semiquinone stabilization in DF bundles, a minimalist model representing active sites of more complex natural diiron and dimanganese proteins. Coordination sphere was modified in the original DF single chain version to incorporate a 2-His-1-carboxylate facial triad, a single metal binding motif, mirroring morphology of mononuclear non-heme enzymes. We discovered breaking symmetry of the metal coordination site leads to a stable construct with one site exhibiting tight affinity for metal, and this single metal binding construct Uno Ferro single chain stabilizes semiquinone radical anions equally efficient as a two-metal binding version. We envision a robust and easy to modify DFsc and UFsc family of proteins would be versatile tools for gaining mechanistic insights of metalloenzymes. One application of DF constructs we\u27ve pursued is to create a hydrolase bridging a heteronuclear metallosite. Phosphoester substrates were introduced to DFsc and UFsc to characterize hydrolase features. Defining the structural and functional properties of hydrolases in DF proteins would facilitate creation of catalysts for bioremediation purposes. We further apply minimalist design approaches to redesign natural proteins displaying no inherent catalytic properties (or showing marginal activity) for a chosen chemical transformation. Calmodulin and myoglobin were selected as model proteins to introduce enzymatic properties rationally designed using non-rigorous set of computational tools to modify the substrate pocket for evolution. We use HSQC-NMR to guide the directed evolution process to arrive at beneficial mutations which enhance reactivity of generated enzymes. Our minimalist design approach also demonstrates the practical application of our calmodulin designs to reveal an acid-base promoted catalysis in conversion of therapeutics to their metabolically active forms. Only a single precisely positioned amino acid is needed to promote conversion of an antirheumatic prodrug leflunomide bearing an isoxazole ring for proton abstraction to the active form teriflunomide. Furthermore, we dissected possible defense mechanisms microorganisms utilize to survive habitats under oxidative stress. A reported diiron catalase conserved exclusively in Mycobacterium tuberculosis (Mtb) was characterized to determine if the protein acts as a dimanganese catalase as we observed the coordination ligands are similar to nonheme catalases. Therefore, we sought to identify metal preference of this four-helix bundle and establish possible mechanisms utilized by Mtb to disproportionate toxic oxygen metabolites. Conversely, a possible defense strategy by humans to promote bacterial clearance in cases where invasive bacteria trigger human innate immune responses was examined. Hemoglobin is found to be antimicrobial generating cytotoxic radicals to sustain microbicidal action. We considered myoglobin involved in similar peroxidatic processes could be reactive in presence of pathogen associated molecular patterns, specifically lipopolysaccharides (LPS), to produce radical cations. Hence, myoglobin was characterized in a mixture of different proteases and LPS for peroxidase activation. Lastly, design of antimicrobial hydrogels is described. Our hydrogel design is based on self-assembly of peptides induced by silver or copper complexation and does not incorporate permanent crosslinking agents. Short peptides of alternating polar and nonpolar amino acids affixed by an unnatural amino acid pyridyl alanine which coordinate metal form a strong hydrogel. The hydrogel is self-healing and can be delivered in syringe format for application in wound healing therapy

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

Engineering Oxygen Reactivity in Heme-Protein Maquettes

Fundamental questions of protein cofactor oxygen reactivity are left unanswered even after years of research due to the limitations imposed by the complexity of natural systems. Natural electron transport proteins are often large with many spectrally overlapping cofactors and fragile to mutation due to multiple roles of each amino acid. The study of the products oxygen reduction is further made difficult by inadequate available methods for kinetic resolution with quantitative yields of different reactive oxygen species (ROS). To first examine how proteins can avoid oxygen reduction for oxygen transport, as in natural globins, an oxygen transporter is designed from first principles of protein folding with each intermediate characterized. This is the first example of a transparently-crafted protein design functioning at natural rates. The ease of this design process, only three steps, and the minimalism of the design, only 8 different amino acids, calls into question many assumptions of what is necessary for a functional protein. This work suggests that functional success is easier reached by modification of gross properties of proteins rather than attempting structural exactness. This work may also aid future designs without necessitating a return to first principles because it not only produces a functional design but a toolbox from which to engineer. Oxygen reactivity was further studied through the development of a system for differentiating the rates and yields of superoxide, hydrogen peroxide and hydroxyl radicals. A series of man-made proteins were tested for oxygen binding, each aforementioned ROS production, and ligand exchange rates to uncover differences in ROS species production and mechanisms. It was found that it takes a single repetitive mutation to design from stable oxygen binder to a rapid outer-sphere superoxide generator. Further, another small set of changes confer preferential hydrogen peroxide generation without a superoxide intermediate through an inner-sphere mechanism. The ability to elucidate mechanistic details of highly homologous proteins and resolve each species of ROS allows us to learn about protein oxygen chemistry and apply this knowledge to the interpretation of data from natural systems, as well as use this knowledge to engineer forward towards industrially- and medically-applicable designer proteins

Building and testing a cognitive approach to the calculus using interactive computer graphics

This thesis consists of a theoretical building of a cognitive approach to the calculus and an empirical testing of the theory in the classroom. A cognitive approach to the teaching of a knowledge domain is defined to be one that aims to make the material potentially meaningful at every stage (in the sense of Ausubel). As a resource in such an approach, the notion of a generic organiser is introduced (after Dienes), which is an environment enabling the learner to explore examples of mathematical processes and concepts, providing cognitive experience to assist in the abstraction of higher order concepts embodied by the organiser. This allows the learner to build and test concepts in a mode 1 environment (in the sense of Skemp) rather than the more abstract modes of thinking typical in higher mathematics. The major hypothesis of the thesis is that appropriately designed generic organisers, supported by an appropriate learning environment, are able to provide students with global gestalts for mathematical processes and concepts at an earlier stage than occurs with current teaching methods. The building of the theory involves an in-depth study of cognitive development, of the cultural growth and theoretical content of the mathematics, followed by the design and programming of appropriate organisers for the teaching of the calculus. Generic organisers were designed for differentiation (gradient of a graph), integration (area), and differential equations, to be coherent ends in themselves as well as laying foundations for the formal theories of both standard and non-standard analysis. The testing is concerned with the program GRADIENT, which is designed to give a global gestalt of the dynamic concept of the gradient of a graph. Three experimental classes (one taught by the researcher in conjunction with the regular class teacher) used the software as an adjunct to the normal study of the calculus and five other classes acted as controls. Matched pairs were selected on a pre-test for the purpose of statistical comparison of performance on the post-test. Data was also collected from a third school where the organisers functioned less well, and from university mathematics students who had not used a computer. The generic organiser GRADIENT, supported by appropriate teaching, enabled the experimental students to gain a global gestalt of the gradient concept. They were able to sketch derivatives. for given graphs significantly better than the controls on the post-test, at a level comparable with more able students reading mathematics at university. Their conceptualizations of gradient and tangent transferred to a new situation involving functions given by different formulae on either side of the point in question, performing significantly better than the control students and at least as well, or better, than those at university