Search

Phytoplankton are key components of the oceanic carbon and sulfur cycles1. During bloom events, some species can emit large amounts of the organosulfur volatile dimethyl sulfide (DMS) into the ocean and consequently the atmosphere, where it can modulate aerosol formation and affect climate2,3. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic interactions. Yet, its role in microbial predator–prey interactions remains elusive with contradicting evidence for its role in either algal chemical defence or in the chemo-attraction of grazers to prey cells4,5. Here we investigated the signalling role of DMS during zooplankton–algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme dimethylsulfoniopropionate lyase (DL) in the bloom-forming alga Emiliania huxleyi6. We inhibited DL activity in E. huxleyi cells in vivo using the selective DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate7 and overexpressed the DL-encoding gene in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as an anti-grazing chemical defence but paradoxically enhanced predation by the grazer Oxyrrhis marina and other microzooplankton and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina growth. Overall, our results demonstrate that DMS-mediated grazing may be ecologically important and prevalent during prey–predator dynamics in aquatic ecosystems. The role of algal DMS revealed here, acting as an eat-me signal for grazers, raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean. Algal production of dimethyl sulfide plays a role in attracting predators and enhancing predation by zooplankton, thus mediating predator–prey relationships in the ocean.

Among the enzyme lineages that undoubtedly emerged prior to the last universal common ancestor is the so-called HUP, which includes Class I aminoacyl tRNA synthetases (AARSs) as well as enzymes mediating NAD, FAD, and CoA biosynthesis. Here, we provide a detailed analysis of HUP evolution, from emergence to structural and functional diversification. The HUP is a nucleotide binding domain that uniquely catalyzes adenylation via the release of pyrophosphate. In contrast to other ancient nucleotide binding domains with the αβα sandwich architecture, such as P-loop NTPases, the HUP’s most conserved feature is not phosphate binding, but rather ribose binding by backbone interactions to the tips of β1 and/or β4. Indeed, the HUP exhibits unusual evolutionary plasticity and, while ribose binding is conserved, the location and mode of binding to the base and phosphate moieties of the nucleotide, and to the substrate(s) reacting with it, have diverged with time, foremost along the emergence of the AARSs. The HUP also beautifully demonstrates how a well- packed scaffold combined with evolvable surface elements promotes evolutionary innovation. Finally, we offer a scenario for the emergence of the HUP from a seed βαβ fragment, and suggest that despite an identical architecture, the HUP and the Rossmann represent independent emergences.

Superoxide dismutases (SODs) are critical metalloenzymes mitigating the damages of the modern oxygenated world. However, the emergence of one family of SODs, the Fe/Mn SOD, has been recurrently proposed to predate the great oxygenation event (GOE). This ancient family lacks metal binding selectivity, but displays strong catalytic selectivity. Therefore, some homologues would only be active when bound to Fe or Mn, although others, dubbed cambialistic, would function when loaded with either ion. This posed the longstanding question about the identity of the cognate metal ion of the first SODs to emerge. In this work, we utilize ancestral sequence reconstruction techniques to infer the earliest SODs. We show that the "ancestors" are active in vivo and in vitro. Further, we test their metal specificity and demonstrate that they are cambialistic in nature. Our findings shed light on how the predicted Last Common Universal Ancestor was capable of dealing with decomposition of the superoxide anion, and the early relationship between life, oxygen, and metal ion availability.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Enzymes are well known for their catalytic abilities, some even reaching "catalytic perfection" in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate-handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a "substrate-handle-binding-first" mechanism ("binding-first" for brevity) instead of "chemistry-first" and we are, therefore, left to wonder what the role of non-catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back-to-back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as "uniform binding" (parallel stabilization of all enzyme-bound states to the same degree). Indeed, we show that a uniform-binding proto-catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate-handle-binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of "binding-first" enzyme families like the aldolase superfamily.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance (EPR) spectroscopy to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif reveals the presence of dimers, even in the absence of RNA, and transient α-helical character. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. The distance distributions between spin labels are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

How protein translation evolved from a simple beginning to its complex and accurate contemporary state is unknown. Aminoacyl-tRNA synthetases (AARSs) define the genetic code by activating amino acids and loading them onto cognate tRNAs. As such, their evolutionary history can shed light on early translation. Using structure-based alignments of the conserved core of Class I AARSs, we reconstructed their phylogenetic tree and ancestral states. Unexpectedly, AARSs charging amino acids that are assumed to have emerged later – such as TrpRS and TyrRS or LysRS and CysRS – appear as the earliest splits in the tree; conversely, those AARSs charging abiotic, early-emerging amino acids, e.g. ValRS, seem to have diverged most recently. Furthermore, the inferred Class I ancestor (excluding TrpRS and TyrRS) lacks the residues that mediate selectivity in contemporary AARSs, and appears to be a generalist that could charge a wide range of amino acids. This ancestor subsequently diverged to two clades: “charged” (which gave rise to ArgRS, GluRS, and GlnRS) and “hydrophobics”, which includes CysRS and LysRS as its outgroups. The ancestors of both clades maintain a wide-accepting pocket that could readily diverge to the contemporary, specialized families. Overall, our findings suggest a “generalist-maintaining” model of class I AARS evolution, in which early statistical translation was kept active by a generalist AARS while the evolution of a specialized, accurate translation system took place.SignificanceAminoacyl-tRNA synthetases (AARS) define the genetic code by linking amino acids with their cognate tRNAs. While contemporary AARSs leverage exquisite molecular recognition and proofreading to ensure translational fidelity, early translation was likely less stringent and operated on a different pool of amino acids. The co-emergence of translational fidelity and the amino acid alphabet, however, is poorly understood. By inferring the evolutionary history of Class I AARSs we found seemingly conflicting signals: Namely, the oldest AARSs apparently operate on the youngest amino acids. We also observed that the early ancestors had broad amino acid specificities, consistent with a model of statistical translation. Our data suggests that a generalist AARS was actively maintained until complete specialization, thereby resolving the age paradox.

Oligomeric proteins are central to cellular life and the duplication and divergence of their genes is a key driver of evolutionary innovations. The duplication of a gene coding for an oligomeric protein has numerous possible outcomes, which motivates questions on the relationship between structural and functional divergence. How do protein oligomeric states diversify after gene duplication? In the simple case of duplication of a homo-oligomeric protein gene, what properties can influence the fate of descendant paralogs toward forming independent homomers or maintaining their interaction as a complex? Furthermore, how are functional innovations associated with the diversification of oligomeric states? Here, we review recent literature and present specific examples in an attempt to illustrate and answer these questions.

Production of molecular oxygen was a turning point in the Earth’s history. The geological record indicates the Great Oxidation Event, which marked a permanent transition to an oxidizing atmosphere around 2.4 Ga. However, the degree to which oxygen was available to life before oxygenation of the atmosphere remains unknown. Here, phylogenetic analysis of all known oxygen-utilizing and -producing enzymes (O2-enzymes) indicates that oxygen became widely available to living organisms well before the Great Oxidation Event. About 60% of the O2-enzyme families whose birth can be dated appear to have emerged at the separation of terrestrial and marine bacteria (22 families, compared to two families assigned to the last universal common ancestor). This node, dubbed the last universal oxygen ancestor, coincides with a burst of emergence of both oxygenases and other oxidoreductases, thus suggesting a wider availability of oxygen around 3.1 Ga. Phylogenetic dating of O2-utilizing enzymes indicates a burst of emergence several hundred million years before the Great Oxidation Event.

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil–helix transitions could be the basis of polyamine regulation in contemporary proteins.

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αβα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αβα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.

Aminoacyl‐tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2–3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 105‐fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H‐ bonding with polar active site groups. This ‘hydroxyl paradox’ is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post‐transfer editing – hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates (‘negative selection’).

Although molecular oxygen is a relative newcomer to the biosphere, it has had a profound impact on metabolism. About 700 oxygen-dependent enzymatic reactions are known, the vast majority of which emerged only after the appearance of oxygen in the biosphere, circa 3 billion years ago. Oxygen was a major driving force for evolutionary innovation-~60% of all known oxygen-dependent enzyme families emerged as such; that is, the founding ancestor was an O

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr., eLife digest Antibiotics treat or prevent infections by killing bacteria or slowing down their growth. A large proportion of these drugs do this by disrupting an essential piece of cellular machinery called the ribosome which the bacteria need to make proteins. However, over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic. Antibiotic resistance is a major threat to global health, and understanding how it emerges and spreads is an important area of research. Recent studies have discovered populations of resistant bacteria carrying a gene for a protein named chloramphenicol-florfenicol resistance, or Cfr for short. Cfr inserts a small modification in to the ribosome that prevents antibiotics from inhibiting the production of proteins, making them ineffective against the infection. To date, Cfr has been found to cause resistance to eight different classes of antibiotics. Identifying which mutations enhance its activity and protect bacteria is vital for designing strategies that fight antibiotic resistance. To investigate how the gene for Cfr could mutate and make bacteria more resistant, Tsai et al. performed a laboratory technique called directed evolution, a cyclic process which mimics natural selection. Genetic changes were randomly introduced in the gene for the Cfr protein and bacteria carrying these mutations were treated with tiamulin, an antibiotic rendered ineffective by the modification Cfr introduces into the ribosome. Bacteria that survived were then selected and had more mutations inserted. By repeating this process several times, Tsai et al. identified ‘super’ variants of the Cfr protein that lead to greater resistance. The experiments showed that these variants boosted resistance by increasing the proportion of ribosomes that contained the protective modification. This process was facilitated by mutations that enabled higher levels of Cfr protein to accumulate in the cell. In addition, the current study allowed, for the first time, direct visualization of how the Cfr modification disrupts the effect antibiotics have on the ribosome. These findings will make it easier for clinics to look out for bacteria that carry these ‘super’ resistant mutations. They could also help researchers design a new generation of antibiotics that can overcome resistance caused by the Cfr protein.

The advent of laboratory directed evolution yielded a fruitful crosstalk between the disciplines of molecular evolution and bio-engineering. Here, we outline recent developments in both disciplines with respect to how one can identify the best starting points for directed evolution, such that highly efficient and robust tailor-made enzymes can be obtained with minimal optimization. Directed evolution studies have highlighted essential features of engineer-able enzymes: highly stable, mutationally robust enzymes with the capacity to accept a broad range of substrates. Robust, evolvable enzymes can be inferred from the natural sequence record. Broad substrate spectrum relates to conformational plasticity and can also be predicted by phylogenetic analyses and/or by computational design. Overall, an increasingly powerful toolkit is becoming available for identifying optimal starting points including network analyses of enzyme superfamilies and other bioinformatics methods.

Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.One sentence summaryWidespread utilization of diverse organophosphate pollutants by over 20 marine bacterial strains represents a new hope for ocean bioremediation.

SummaryMulti-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite rather than sequential reactions (“counter-enzymes”): glutamate synthase (GltAB), and glutamate dehydrogenase (GudB), that make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit GudB’s activity as this enzyme is constitutively expressed including in glutamate-limiting conditions. Using cryo-electron microscopy, we elucidated the structure of the complex and the basis of GudB’s inhibition. Finally, we show that this complex that exhibits unusual oscillatory progress curves is a necessity for planktonic growth in glutamate-limiting conditions, but is also essential for biofilm growth in glutamate-rich media, suggesting a regulatory role at fluctuating glutamate concentrations.

Significance Across the Tree of Life, life’s phenotypic diversity has been accompanied by a massive expansion of the protein universe. Compared with simple prokaryotes that harbor thousands of proteins, plants and animals harbor hundreds of thousands of proteins that are also longer, multidomain, and comprise a variety of folds and fold combinations, repeated segments, and beta-rich architectures that make them prone to misfolding and aggregation. Surprisingly, the relative representation of core chaperones, those dedicated to maintaining the folding quality of these increasingly complex proteomes, did not change from prokaryotic to mammalian genomes. To reconcile the expanding proteomes, core chaperones have rather increased in cellular abundance and evolved to function cooperatively as a network, combined with their supporting workforce, the cochaperones., Across the Tree of Life (ToL), the complexity of proteomes varies widely. Our systematic analysis depicts that from the simplest archaea to mammals, the total number of proteins per proteome expanded ∼200-fold. Individual proteins also became larger, and multidomain proteins expanded ∼50-fold. Apart from duplication and divergence of existing proteins, completely new proteins were born. Along the ToL, the number of different folds expanded ∼5-fold and fold combinations ∼20-fold. Proteins prone to misfolding and aggregation, such as repeat and beta-rich proteins, proliferated ∼600-fold and, accordingly, proteins predicted as aggregation-prone became 6-fold more frequent in mammalian compared with bacterial proteomes. To control the quality of these expanding proteomes, core chaperones, ranging from heat shock proteins 20 (HSP20s) that prevent aggregation to HSP60, HSP70, HSP90, and HSP100 acting as adenosine triphosphate (ATP)-fueled unfolding and refolding machines, also evolved. However, these core chaperones were already available in prokaryotes, and they comprise ∼0.3% of all genes from archaea to mammals. This challenge—roughly the same number of core chaperones supporting a massive expansion of proteomes—was met by 1) elevation of messenger RNA (mRNA) and protein abundances of the ancient generalist core chaperones in the cell, and 2) continuous emergence of new substrate-binding and nucleotide-exchange factor cochaperones that function cooperatively with core chaperones as a network.

Oxygen is a major metabolic driving force that enabled the expansion of metabolic networks including new metabolites and new enzymes. It had a dramatic impact on the primary electron transport chain where it serves as terminal electron acceptor, but oxygen is also used by many enzymes as electron acceptor for a variety of reactions. The organismal oxygen phenotype, aerobic vs. anaerobic, should be manifested in its O2-utilizing enzymes. Traditionally, enzymes involved in primary oxygen metabolism such as cytochrome c, and reactive oxygen species (ROS)-neutralizing enzymes (e.g. catalase), were used as identifiers of oxygen phenotype. However, these enzymes are often found in strict anaerobes. We aimed to identify the O2-utilizing enzymes that may distinguish between aerobes and anaerobes. To this end, we annotated the O2-utilizing enzymes across the prokaryotic tree of life. We recovered over 700 enzymes and mapped their presence/absence in 272 representative genomes. As seen before, enzymes mediating primary oxygen metabolism, and ROS neutralizing enzymes, could be found in both aerobes and anaerobes. However, there exists a subset of enzymes, primarily oxidases that catabolyze various substrates, including amino acids and xenobiotics, that are preferentially enriched in aerobes. Overall it appears that the total number of oxygen-utilizing enzymes, and the presence of enzymes involved in 'peripheral', secondary oxygen metabolism, can reliably distinguish aerobes from anaerobes based solely on genome sequences. These criteria can also indicate the oxygen phenotype in metagenomic samples.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple βα repeat proteins bind NTPs, but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one β-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes comprise a plausible description of the sequence, structure and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.Significance statementIt is widely assumed that today’s large and complex proteins emerged from much shorter and simpler polypeptides. Yet the nature of these early precursors remains enigmatic. We describe polypeptides that contain one of the earliest protein motifs, a phosphate-binding loop, or P-loop, embedded in a single beta-alpha element. These P-loop prototypes show intriguing characteristics of a primordial world comprised of nucleic acids and peptides. They are ‘generalists’ capable of binding different phospho-ligands, including inorganic polyphosphates and single-stranded DNA. Nonetheless, in promoting double-stranded DNA unwinding and strand-exchange they resemble modern P-loop helicases and recombinases. Our study describes a missing link in the evolution of complex proteins – simple polypeptides that tangibly relate to contemporary P-loop enzymes in sequence, structure and function.

The vast majority of theoretically possible polypeptide chains do not fold, let alone confer function. Hence, protein evolution from preexisting building blocks has clear potential advantages over ab initio emergence from random sequences. In support of this view, sequence similarities between different proteins is generally indicative of common ancestry, and we collectively refer to such homologous sequences as ‘themes’. At the domain level, sequence homology is routinely detected. However, short themes which are segments, or fragments of intact domains, are particularly interesting because they may provide hints about the emergence of domains, as opposed to divergence of preexisting domains, or their mixing-and-matching to form multi-domain proteins. Here we identified 525 representative short themes, comprising 20-to-80 residues, that are unexpectedly shared between domains considered to have emerged independently. Among these ‘bridging themes’ are ones shared between the most ancient domains, e.g., Rossmann, P-loop NTPase, TIM-barrel, Flavodoxin, and Ferredoxin-like. We elaborate on several particularly interesting cases, where the bridging themes mediate ligand binding. Ligand binding may have contributed to the stability and the plasticity of these building blocks, and to their ability to invade preexisting domains or serve as starting points for completely new domains.

Phytoplankton are key components of the oceanic carbon and sulfur cycles 1. During bloom events, some species can emit massive amounts of the organosulfur volatile dimethyl sulfide (DMS) to the atmosphere, where it can modulate aerosol formation and affect climate. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic-level interactions. Yet its role in microbial predator-prey interactions remains elusive with contradicting evidence for its role in algal chemical defense and in grazer’s chemoattraction to prey cells. Here, we investigated the signaling role of DMS during zooplankton-algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme (Dimethylsulfoniopropionate lyase, DL) from the bloom-forming alga Emiliania huxleyi. We inhibited DL activity in live E. huxleyi cells by the novel DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate (Br-DMSP) , and overexpressed DL in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as anti-grazing chemical defense, and paradoxically enhanced grazing by the model microzooplankton Oxyrrhis marina and other micro- and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina’s growth. Overall, our results demonstrate that DMS-mediated herbivory may be ecologically important and prevalent during prey-predator dynamics in oceanic ecosystems. The role of DMS as an appetizing signal to grazers revealed here raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean.

Dating back to the last universal common ancestor (LUCA), the P-loop NTPases and Rossmanns now comprise the most ubiquitous and diverse enzyme lineages. Intriguing similarities in their overall architecture and phosphate binding motifs suggest common ancestry; however, due to a lack of sequence identity and some fundamental structural differences, these families are considered independent emergences. To address this longstanding dichotomy, we systematically searched for ‘bridge proteins’ with structure and sequence elements shared by both lineages. We detected homologous segments that span the first βαβ segment of both lineages and include two key functional motifs: (i) a phosphate binding loop – the ‘Walker A’ motif of P-loop NTPases or the Rossmann equivalent, both residing at the N-terminus of α1; and (ii) an Asp at the tip of β2. The latter comprises the ‘Walker B’ aspartate that chelates the catalytic metal in P-loop NTPases, or the canonical Rossmann β2-Asp that binds the cofactor’s ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the β2-Asp to take either one of these two roles. We conclude that common P-loops/Rossmann ancestry is plausible, although convergence cannot be completely ruled out. Regardless, both lineages most likely emerged from a polypeptide comprising a βαβ segment carrying the above two functional motifs, a segment that comprises the core of both enzyme families to this very day.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remains unclear. Further, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein to glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyper-acidic protein at sub-millimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+and Ca2+had an effect similar to a di-amine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues, and that (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

Oligomeric proteins are central to life. Duplication and divergence of their genes is a key evolutionary driver, also because duplications can yield very different outcomes. Given a homomeric ancestor, duplication can yield two paralogs that form two distinct homomeric complexes, or a heteromeric complex comprising both paralogs. Alternatively, one paralog remains a homomer while the other acquires a new partner. However, so far, conflicting trends have been noted with respect to which fate dominates, primarily because different methods and criteria are being used to assign the interaction status of paralogs. Here, we systematically analyzed all Saccharomyces cerevisiae and Escherichia coli oligomeric complexes that include paralogous proteins. We found that the proportions of homo-hetero duplication fates strongly depend on a variety of factors, yet that nonetheless, rigorous filtering gives a consistent picture. In E. coli about 50%, of the paralogous pairs appear to have retained the ancestral homomeric interaction, whereas in S. cerevisiae only ~10% retained a homomeric state. This difference was also observed when unique complexes were counted instead of paralogous gene pairs. We further show that this difference is accounted for by multiple cases of heteromeric yeast complexes that share common ancestry with homomeric bacterial complexes. Our analysis settles contradicting trends and conflicting previous analyses, and provides a systematic and rigorous pipeline for delineating the fate of duplicated oligomers in any organism for which protein-protein interaction data are available., Author summary About half of all proteins assemble as oligomers, either by self-interaction (homomers) or via interaction with another protein (heteromers). The latter can be unrelated, yet, quite commonly, the interacting proteins are paralogs, namely two genes that arose by gene duplication. Indeed, while a homomer is encoded by a single gene, heteromers demand two genes as a minimum. Duplication can therefore yield two discrete homomeric complexes or a single heteromer. Do paralogs tend to retain the ancestral homomeric interaction, or do they mostly diverge into heteromeric complexes? Despite several studies addressing this question, to date, we lack a systematic, rigorous approach for delineating the oligomeric fates of paralogs on an organism scale. To this end, we developed a new pipeline for analysis of molecular interaction databases that includes various filtering steps and unambiguous definitions of all possible oligomeric fates. Applying this method to Escherichia coli and Saccharomyces cerevisiae we noted that paralogous pairs tend to remain homomeric in the former while in the latter heteromeric complexes dominate. We consequently note a systematic trend of homomeric bacterial proteins diverging into heteromeric complexes in eukaryotes.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

Production of molecular oxygen was a turning point in the Earth's history. The geological record indicates the Great Oxidation Event, which marked a permanent transition to an oxidizing atmosphere around 2.4 Ga. However, the degree to which oxygen was available to life before oxygenation of the atmosphere remains unknown. Here, phylogenetic analysis of all known oxygen-utilizing and -producing enzymes (O

This Special Issue is composed of 10 reviews that delve into the intricacies behind enzyme promiscuity and evolution, an area that is of increasing interest in the biological research community. In particular, the reviews in this Special Issue explore enzyme promiscuity and evolution in the context of cellular metabolism, as discussed in this introductory Editorial. It is our hope that you enjoy these fascinating and informative reviews and we wish to thank the authors for their compelling contributions to The FEBS Journal. doi: 10.1111/febs.12650.

Natural product biosynthesis (NPB) is the Panda's Thumb of evolutionary biochemistry. Arm races between organisms, and ever-changing environments, result in relentless innovation. This review focusses on enzyme evolution in NPB. First, we review cases of de novo emergence, whereby a completely new enzymatic activity arose in a ligand-binding protein, or a new enzyme emerged including a completely new scaffold. Second, we briefly review the current models for enzyme evolution, and how they explain the inherent promiscuity of NPB enzymes and their tendency to produce multiple related products. We thus suggest that NPB enzymes a priori evolved to generate a specific product; they are, however, trapped in a multifunctional, generalist evolutionary state and thereby produce a diversity of products.

De novo emergence, and emergence of the earliest proteins specifically, demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can peptides confer evolutionary relevant functions, let alone with minimal abiotic amino acid alphabets? How can such polypeptides evolve into mature proteins? Specifically, while nucleic acids binding is presumed a primordial function, it demands basic amino acids that do not readily form abiotically. To address these questions, we describe an experimentally-validated trajectory from a phase-separating polypeptide to a dsDNA-binding protein. The intermediates comprise sequence-duplicated, functional proteins made of only 10 amino acid types, with ornithine, which can form abiotically, as the only basic amino acid. Statistical, chemical modification of ornithine sidechains to arginine promoted structure and function. The function concomitantly evolved – from phase separation with RNA (coacervates) to avid and specific dsDNA binding – thereby demonstrating a smooth, gradual peptide-to-protein transition with respect to sequence, structure, and function.

Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.