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 protein-mediated 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.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three ß-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest ß-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

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 ß-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 (kcat/KM~2·105 M-1 s-1, kcat~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.

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases.

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 ß-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 ß-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.

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.

Equilibrium partially folded states of B. licheniformis[Formula: see text]-lactamase.

[Formula: see text]-Lactamases (penicillinases) facilitate bacterial resistance to antibiotics and are excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A [Formula: see text]-lactamase with three tryptophan residues located one in each of its two domains and one in the interface between domains. The conformational landscape of three well-characterized ESP Trp[Formula: see text]Phe mutants was characterized in equilibrium unfolding experiments by measuring tryptophan fluorescence, far-UV CD, activity, hydrodynamic radius, and limited proteolysis. The Trp[Formula: see text]Phe substitutions had little impact on the native conformation, but changed the properties of the partially folded states populated at equilibrium. The results were interpreted in the framework of modern theories of protein folding.

Engineering ancestral protein hyperstability.

Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach.

Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme.

ß-Lactamases are produced by many modern bacteria as a mechanism of resistance toward ß-lactam antibiotics, the most common antibiotics in use. ß-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A ß-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern ß-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant ß-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient ß-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and ß-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins.

Consensus-sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However, the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood. Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of extant, class A ß-lactamases, as compared with the properties of ancient Precambrian ß-lactamases resurrected in the laboratory. These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although to a lower extent than ancient ß-lactamases. These results: (i) highlight the phenotypic differences between consensus variants and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation of protein variants having large numbers of mutational changes while possessing unique biomolecular properties.

Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian ß-lactamases.

We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A ß-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old ß-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern ß-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old ß-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.

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 protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time 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.

Host diversification is concurrent with linear motif evolution in a Mastadenovirus hub protein.

Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein-protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein-protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.

A protocol to study bacteriophage adaptation to new hosts.

A general protocol for the experimental assessment of bacteriophage adaptation to new hosts is described. We use as a model system the lytic phage T7 and an engineered E. coli strain modified to hamper the recruitment of a known proviral factor. Our protocol includes steps of phage amplification, plaque and liquid lysis assays, and DNA extraction for next-generation sequencing of the viral genome over several rounds of laboratory evolution thus allowing the investigation of the sequence determinants of viral adaptation. For complete information on the generation and use of this protocol, please refer to Luzon-Hidalgo et al. (2021).

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

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.

Evidence for a role of phenotypic mutations in virus adaptation.

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 recruitment was enabled by phenotypic mutations 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 phenotypic mutations may generally contribute to the capability of viruses to evade antiviral strategies.

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

TEM-1 ß-lactamase degrades ß-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of ß-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 ß-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 ß-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.

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase.

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.