Introduction to the Special Issue on Early Evolution and the Last Common Ancestor.

The early evolution of life spans an extensive period preceding the emergence of the first eukaryotic cell. This epoch, which transpired from 4.5 to 2.5 billion years ago, marked the advent of many fundamental cellular attributes and witnessed the existence of the Last Common Ancestor (LCA) of all life forms. Uncovering and reconstructing this elusive LCA's characteristics and genetic makeup represents a formidable challenge and a pivotal pursuit in early evolution. While most scientific accounts concur that the LCA resembles contemporary prokaryotes, its precise definition, genome composition, metabolic capabilities, and ecological niche remain subjects of contentious debate.

RNA or DNA? Revisiting the Chemical Nature of the Cenancestral Genome.

One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome.

The Fate of Duplicated Enzymes in Prokaryotes: The Case of Isomerases.

The isomerases are a unique enzymatic class of enzymes that carry out a great diversity of chemical reactions at the intramolecular level. This class comprises about 300 members, most of which are involved in carbohydrate and terpenoid/polyketide metabolism. Along with oxidoreductases and translocases, isomerases are one of the classes with the highest ratio of paralogous enzymes. Due to its relatively small number of members, it is plausible to explore it in greater detail to identify specific cases of gene duplication. Here, we present an analysis at the level of individual isomerases and identify different members that seem to be involved in duplication events in prokaryotes. As was suggested in a previous study, there is no homogeneous distribution of paralogs, but rather they accumulate into a few subcategories, some of which differ between Archaea and Bacteria. As expected, the metabolic processes with more paralogous isomerases have to do with carbohydrate metabolism but also with RNA modification (a particular case involving an rRNA-modifying isomerase is thoroughly discussed and analyzed in detail). Overall, our findings suggest that the most common fate for paralogous enzymes is the retention of the original enzymatic function, either associated with a dosage effect or with differential expression in response to changing environments, followed by subfunctionalization and, to a much lesser degree, neofunctionalization, which is consistent with what has been reported elsewhere.

Structural Analysis of Monomeric RNA-Dependent Polymerases Revisited.

In the past few years, our understanding of the RNA virosphere has changed dramatically due to the growth and spurt of metagenomics, exponentially increasing the number of RNA viral sequences, and providing a better understanding of their range of potential hosts. As of today, the only conserved protein among RNA viruses appears to be the monomeric RNA-dependent RNA polymerase. This enzyme belongs to the right-hand DNA-and RNA polymerases, which also includes reverse transcriptases and eukaryotic replicative DNA polymerases. The ubiquity of this protein in RNA viruses makes it a unique evolutionary marker and an appealing broad-spectrum antiviral target. In this work pairwise structural comparisons of viral RdRps and RTs were performed, including tertiary structures that have been obtained in the last few years. The resulting phylogenetic tree shows that the RdRps from (+)ss- and dsRNA viruses might have been recruited several times throughout the evolution of mobile genetic elements. RTs also display multiple evolutionary routes. We have identified a structural core comprising the entire palm, a large moiety of the fingers and the N-terminal helices of the thumb domain, comprising over 300 conserved residues, including two regions that we have named the "knuckles" and the "hypothenar eminence". The conservation of an helix bundle in the region preceding the polymerase domain confirms that (-)ss and dsRNA Reoviruses' polymerases share a recent ancestor. Finally, the inclusion of DNA polymerases into our structural analyses suggests that monomeric RNA-dependent polymerases might have diverged from B-family polymerases.

Reconstructing the Last Common Ancestor: Epistemological and Empirical Challenges.

Reconstructing the genetic traits of the Last Common Ancestor (LCA) and the Tree of Life (TOL) are two examples of the reaches of contemporary molecular phylogenetics. Nevertheless, the whole enterprise has led to paradoxical results. The presence of Lateral Gene Transfer poses epistemic and empirical challenges to meet these goals; the discussion around this subject has been enriched by arguments from philosophers and historians of science. At the same time, a few but influential research groups have aimed to reconstruct the LCA with rich-in-detail hypotheses and high-resolution gene catalogs and metabolic traits. We argue that LGT poses insurmountable challenges for detailed and rich in details reconstructions and propose, instead, a middle-ground position with the reconstruction of a slim LCA based on traits under strong pressures of Negative Natural Selection, and for the need of consilience with evidence from organismal biology and geochemistry. We defend a cautionary perspective that goes beyond the statistical analysis of gene similarities and assumes the broader consequences of evolving empirical data and epistemic pluralism in the reconstruction of early life.

The Semi-Enzymatic Origin of Metabolic Pathways: Inferring a Very Early Stage of the Evolution of Life.

The early evolution of life is a period with many important events with a lot of big and open questions. One of them is the evolution of metabolic pathways, which means the origin and assembly of enzymes that act together. The retrograde hypothesis was the first attempt to explain the origin and evolutionary history of metabolic pathways; Norman Horowitz developed this first significant hypothesis. This idea was followed by relevant proposals developed by Sam Granick, who proposed the "forward direction hypothesis," and then the successful idea of "Patchwork" assembly proposed independently by Martynas Ycas and Roy Jensen. Since then, a few new hypotheses were proposed; one of the most influential was made by Antonio Lazcano and Stanley Miller in the Journal Molecular Evolution, the "semi-enzymatic origin" of metabolic pathways. This article was cited more than 160 times, including in most papers published about the early evolution of metabolism, placing it as influential work in the field. The ideas proposed in this work and their effects on studying the origin and early evolution of life are analyzed.

Repetitive DNA profile of the amphibian mitogenome.

BACKGROUND: Repetitive DNA elements such as direct and inverted repeat sequences are present in every genome, playing numerous biological roles. In amphibians, the functions and effects of the repeat sequences have not been extensively explored. We consider that the data of mitochondrial genomes in the NCBI database are a valuable alternative to generate a better understanding of the molecular dynamic of the repeat sequences in the amphibians. RESULTS: This work presents the development of a strategy to identify and quantify the total amount of repeat sequences with lengths from 5 to 30 base pairs in the amphibian mitogenomes. The results show differences in the abundance of repeat sequences among amphibians and bias to specific genomic regions that are not easily associated with the classical amphibian ancestry. CONCLUSIONS: Derived from these analyses, we show that great variability of the repeat sequences exists among amphibians, demonstrating that the mitogenomes of these organisms are dynamic.

Alarmones as Vestiges of a Bygone RNA World.

All known alarmones are ribonucleotides or ribonucleotide derivatives that are synthesized when cells are under stress conditions, triggering a stringent response that affects major processes such as replication, gene expression, and metabolism. The ample phylogenetic distribution of alarmones (e.g., cAMP, Ap(n)A, cGMP, AICAR, and ZTP) suggests that they are very ancient molecules that may have already been present in cellular systems prior to the evolutionary divergence of the Archaea, Bacteria, and Eukarya domains. Their chemical structure, wide biological distribution, and functional role in highly conserved cellular processes support the possibility that these modified nucleotides are molecular fossils of an epoch in the evolution of chemical signaling and metabolite sensing during which RNA molecules played a much more conspicuous role in biological catalysis and genetic information.

On the Early Evolution of Catabolic Pathways: A Comparative Genomics Approach. I. The Cases of Glucose, Ribose, and the Nucleobases Catabolic Routes.

Compared with the large corpus of published work devoted to the study of the origin and early development of anabolism, little attention has been given to the discussion of the early evolution of catabolism in spite of its significance. In the present study, we have used comparative genomics to explore the evolution and phylogenetic distribution of the enzymes that catalyze the extant catabolic pathways of the monosaccharides glucose and ribose, as well as those of the nucleobases adenine, guanine, cytosine, uracil, and thymine. Based on the oxygen dependence of the enzymes, their conservation, and evolution, we speculate on the relative antiquity of the pathways. Our results allow us to suggest which catabolic pathways and enzymes may have already been present in the last common ancestor. We conclude that the enzymatic degradations of ribose, as well as those of purines adenine and guanine, are among the most ancient catabolic pathways which can be traced by protein-based methodologies.

Methanogenesis on Early Stages of Life: Ancient but Not Primordial.

Of the six known autotrophic pathways, the Wood-Ljungdahl pathway (WL) is the only one present in both the acetate producing Bacteria (homoacetogens) and the methane producing Archaea (hydrogenotrophic methanogens), and it has been suggested that WL is one of the oldest metabolic pathways. However, only the so-called carbonyl branch is shared by Archaea and Bacteria, while the methyl branch is different, both in the number of reactions and enzymes, which are not homologous among them. In this work we show that some parts of the methyl branch of archaeal Wood-Ljungdahl pathway (MBWL) are present in bacteria as well as in non-methanogen archaea, although the tangled evolutionary history of MBWL cannot be traced back to the Last Common Ancestor. We have also analyzed the different variants of methanogenesis (hydrogenotrophic, acetoclastic and methylotrophic pathways), and concluded that each of these pathways, and every different enzyme or subunit (in the case of multimeric enzymes), has their own intricate evolutionary history. Our study supports the scenario of hydrogenotrophic methanogenesis being older than the other variants, albeit not old enough to be present in the last archaeal common ancestor.

Can an Imidazole Be Formed from an Alanyl-Seryl-Glycine Tripeptide under Possible Prebiotic Conditions?

The five-membered heterocyclic imidazole group, which is an essential component of purines, histidine and many cofactors, has been abiotically synthesized in different model experiments that attempt to simulate the prebiotic environment. The evolutionary significance of imidazoles is highlighted not only by its presence in nucleic acid components and in histidine, but also by experimental reports of its ability to restore the catalytic activity of ribozymes. However, as of today there are no reports of histidine in carbonaceous chondrites, and although the abiotic synthesis of His reported by Shen et al. (1987, 1990a) proceeds via an Amadori rearrangement, like in the biosynthesis of histidine, neither the reactants nor the conditions are truly prebiotic. Based on the autocatalytic biosynthesis of 4-methylidene-imidazole-one (MIO), a cofactor of some members of the amino acid aromatic ammonia-lyases and aminomutases, which occur via the self-condensation of a simple Ala-Ser-Gly motif within the sequence of the enzymes, we propose a possible prebiotic synthesis of an imidazolide.

Comparative evaluation of bioinformatic tools for virus-host prediction and their application to a highly diverse community in the Cuatro Ciénegas Basin, Mexico.

Due to the enormous diversity of non-culturable viruses, new viruses must be characterized using culture-independent techniques. The associated host is an important phenotypic feature that can be inferred from metagenomic viral contigs thanks to the development of several bioinformatic tools. Here, we compare the performance of recently developed virus-host prediction tools on a dataset of 1,046 virus-host pairs and then apply the best-performing tools to a metagenomic dataset derived from a highly diverse transiently hypersaline site known as the Archaean Domes (AD) within the Cuatro Ciénegas Basin, Coahuila, Mexico. Among host-dependent methods, alignment-based approaches had a precision of 66.07% and a sensitivity of 24.76%, while alignment-free methods had an average precision of 75.7% and a sensitivity of 57.5%. RaFAH, a virus-dependent alignment-based tool, had the best overall performance (F1_score = 95.7%). However, when predicting the host of AD viruses, methods based on public reference databases (such as RaFAH) showed lower inter-method agreement than host-dependent methods run against custom databases constructed from prokaryotes inhabiting AD. Methods based on custom databases also showed the greatest agreement between the source environment and the predicted host taxonomy, habitat, lifestyle, or metabolism. This highlights the value of including custom data when predicting hosts on a highly diverse metagenomic dataset, and suggests that using a combination of methods and qualitative validations related to the source environment and predicted host biology can increase the number of correct predictions. Finally, these predictions suggest that AD viruses infect halophilic archaea as well as a variety of bacteria that may be halophilic, halotolerant, alkaliphilic, thermophilic, oligotrophic, sulfate-reducing, or marine, which is consistent with the specific environment and the known geological and biological evolution of the Cuatro Ciénegas Basin and its microorganisms.

Low complexity regions (LCRs) contribute to the hypervariability of the HIV-1 gp120 protein.

Low complexity regions (LCRs) are sequences of nucleic acids or proteins defined by a compositional bias. Their occurrence has been confirmed in sequences of the three cellular lineages (Bacteria, Archaea and Eucarya), and has also been reported in viral genomes. We present here the results of a detailed computer analysis of the LCRs present in the HIV-1 glycoprotein 120 (gp120) encoded by the viral gene env. The analysis was performed using a sample of 3637 Env polyprotein sequences derived from 4117 completely sequenced and translated HIV-1 genomes available in public databases as of December 2012. We have identified 1229 LCRs located in four different regions of the gp120 protein that correspond to four of the five regions that have been identified as hypervariable (V1, V2, V4 and V5). The remaining 29 LCRs are found in the signal peptide and in the conserved regions C2, C3, C4 and C5. No LCR has been identified in the hypervariable region V3. The LCRs detected in the V1, V2, V4, and V5 hypervariable regions exhibit a high Asn content in their amino acid composition, which very likely correspond to glycosylation sites, which may contribute to the retroviral ability to avoid the immune system. In sharp contrast with what is observed in gp120 proteins lacking LCRs, the glycosylation sites present in LCRs tend to be clustered towards the center of the region forming well-defined islands. The results presented here suggest that LCRs represent a hitherto undescribed source of genomic variability in lentivirus, and that these repeats may represent an important source of antigenic variation in HIV-1 populations. The results reported here may exemplify the evolutionary processes that may have increased the size of primitive cellular RNA genomes and the role of LCRs as a source of raw material during the processes of evolutionary acquisition of new functions.

Norvaline and norleucine may have been more abundant protein components during early stages of cell evolution.

The absence of the hydrophobic norvaline and norleucine in the inventory of protein amino acids is readdressed. The well-documented intracellular accumulation of these two amino acids results from the low-substrate specificity of the branched-chain amino acid biosynthetic enzymes that act over a number of related α-ketoacids. The lack of absolute substrate specificity of leucyl-tRNA synthase leads to a mischarged norvalyl-tRNA(Leu) that evades the translational proofreading activities and produces norvaline-containing proteins, (cf. Apostol et al. J Biol Chem 272:28980-28988, 1997). A similar situation explains the presence of minute but detectable amounts of norleucine in place of methionine. Since with few exceptions both leucine and methionine are rarely found in the catalytic sites of most enzymes, their substitution by norvaline and norleucine, respectively, would have not been strongly hindered in small structurally simple catalytic polypeptides during the early stages of biological evolution. The report that down-shifts of free oxygen lead to high levels of intracellular accumulation of pyruvate and the subsequent biosynthesis of norvaline (Soini et al. Microb Cell Factories 7:30, 2008) demonstrates the biochemical and metabolic consequences of the development of a highly oxidizing environment. The results discussed here also suggest that a broader definition of biomarkers in the search for extraterrestrial life may be required.

Subtle structural differences of nucleotide analogs may impact SARS-CoV-2 RNA-dependent RNA polymerase and exoribonuclease activity.

The rapid spread and public health impact of the novel SARS-CoV-2 variants that cause COVID-19 continue to produce major global impacts and social distress. Several vaccines were developed in record time to prevent and limit the spread of the infection, thus playing a pivotal role in controlling the pandemic. Although the repurposing of available drugs attempts to provide therapies of immediate access against COVID-19, there is still a need for developing specific treatments for this disease. Remdesivir, molnupiravir and Paxlovid remain the only evidence-supported antiviral drugs to treat COVID-19 patients, and only in severe cases. To contribute on the search of potential Covid-19 therapeutic agents, we targeted the viral RNA-dependent RNA polymerase (RdRp) and the exoribonuclease (ExoN) following two strategies. First, we modeled and analyzed nucleoside analogs sofosbuvir, remdesivir, favipiravir, ribavirin, and molnupiravir at three key binding sites on the RdRp-ExoN complex. Second, we curated and virtually screened a database containing 517 nucleotide analogs in the same binding sites. Finally, we characterized key interactions and pharmacophoric features presumably involved in viral replication halting at multiple sites. Our results highlight structural modifications that might lead to more potent SARS-CoV-2 inhibitors against an expansive range of variants and provide a collection of nucleotide analogs useful for screening campaigns.

Two short low complexity regions (LCRs) are hallmark sequences of the Delta SARS-CoV-2 variant spike protein.

Low complexity regions (LCRs) are protein sequences formed by a set of compositionally biased residues. LCRs are extremely abundant in cellular proteins and have also been reported in viruses, where they may partake in evasion of the host immune system. Analyses of 28,231 SARS-CoV-2 whole proteomes and of 261,051 spike protein sequences revealed the presence of four extremely conserved LCRs in the spike protein of several SARS-CoV-2 variants. With the exception of Iota, where it is absent, the Spike LCR-1 is present in the signal peptide of 80.57% of the Delta variant sequences, and in other variants of concern and interest. The Spike LCR-2 is highly prevalent (79.87%) in Iota. Two distinctive LCRs are present in the Delta spike protein. The Delta Spike LCR-3 is present in 99.19% of the analyzed sequences, and the Delta Spike LCR-4 in 98.3% of the same set of proteins. These two LCRs are located in the furin cleavage site and HR1 domain, respectively, and may be considered hallmark traits of the Delta variant. The presence of the medically-important point mutations P681R and D950N in these LCRs, combined with the ubiquity of these regions in the highly contagious Delta variant opens the possibility that they may play a role in its rapid spread.

Metalloproteins and the pyrite-based origin of life: a critical assessment.

We critically examine the proposal by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that putative transition metal binding sites in protein components of the translation machinery of hyperthermophiles provide evidence of a direct relationship with the FeS clusters of pyrite and thus indicate an autotrophic origin of life in volcanic environments. Analysis of completely sequenced cellular genomes of Bacteria, Archaea and Eucarya does not support the suggestion by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that aminoacyl-tRNA synthetases and ribosomal proteins bear sequence signatures typical of strong covalent metal bonding whose absence in mesophilic species reveals a process of adaptation towards less extreme environments.

The Role of Gene Duplication in the Divergence of Enzyme Function: A Comparative Approach.

Gene duplication is a crucial process involved in the appearance of new genes and functions. It is thought to have played a major role in the growth of enzyme families and the expansion of metabolism at the biosphere's dawn and in recent times. Here, we analyzed paralogous enzyme content within each of the seven enzymatic classes for a representative sample of prokaryotes by a comparative approach. We found a high ratio of paralogs for three enzymatic classes: oxidoreductases, isomerases, and translocases, and within each of them, most of the paralogs belong to only a few subclasses. Our results suggest an intricate scenario for the evolution of prokaryotic enzymes, involving different fates for duplicated enzymes fixed in the genome, where around 20-40% of prokaryotic enzymes have paralogs. Intracellular organisms have a lesser ratio of duplicated enzymes, whereas free-living enzymes show the highest ratios. We also found that phylogenetically close phyla and some unrelated but with the same lifestyle share similar genomic and biochemical traits, which ultimately support the idea that gene duplication is associated with environmental adaptation.