Metabolite-Enzyme Coevolution: From Single Enzymes to Metabolic Pathways and Networks.

How individual enzymes evolved is relatively well understood. However, individual enzymes rarely confer a physiological advantage on their own. Judging by its current state, the emergence of metabolism seemingly demanded the simultaneous emergence of many enzymes. Indeed, how multicomponent interlocked systems, like metabolic pathways, evolved is largely an open question. This complexity can be unlocked if we assume that survival of the fittest applies not only to genes and enzymes but also to the metabolites they produce. This review develops our current knowledge of enzyme evolution into a wider hypothesis of pathway and network evolution. We describe the current models for pathway evolution and offer an integrative metabolite-enzyme coevolution hypothesis. Our hypothesis addresses the origins of new metabolites and of new enzymes and the order of their recruitment. We aim to not only survey established knowledge but also present open questions and potential ways of addressing them.

De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of ß-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.

Automated Design of Efficient and Functionally Diverse Enzyme Repertoires.

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.

Enzyme promiscuity: a mechanistic and evolutionary perspective.

Many, if not most, enzymes can promiscuously catalyze reactions, or act on substrates, other than those for which they evolved. Here, we discuss the structural, mechanistic, and evolutionary implications of this manifestation of infidelity of molecular recognition. We define promiscuity and related phenomena and also address their generality and physiological implications. We discuss the mechanistic enzymology of promiscuity--how enzymes, which generally exert exquisite specificity, catalyze other, and sometimes barely related, reactions. Finally, we address the hypothesis that promiscuous enzymatic activities serve as evolutionary starting points and highlight the unique evolutionary features of promiscuous enzyme functions.

Utilization of diverse organophosphorus pollutants by marine bacteria.

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.

The evolutionary history of the HUP domain.

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 αßα 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 ß1 and/or ß4. 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 ßαß fragment, and suggest that despite an identical architecture, the HUP and the Rossmann represent independent emergences.

A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-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 ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

On the evolution of chaperones and cochaperones and the expansion of proteomes across the Tree of Life.

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.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

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 ßα 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 ß-(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 compose 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.

On the Origins of Enzymes: Phosphate-Binding Polypeptides Mediate Phosphoryl Transfer to Synthesize Adenosine Triphosphate.

Reactions involving the transfer of a phosphoryl (-PO32-) group are fundamental to cellular metabolism. These reactions are catalyzed by enzymes, often large and complex, belonging to the phosphate-binding loop (P-loop) nucleoside triphosphatase (NTPase) superfamily. Due to their critical importance in life, it is reasonable to assume that phosphoryl-transfer reactions were also crucial in the pre-LUCA (last universal common ancestor) world and mediated by precursors that were simpler, in terms of their sequence and structure, relative to their modern-day enzyme counterparts. Here, we demonstrate that short phosphate-binding polypeptides (∼50 residues) comprising a single, ancestrally inferred, P-loop or Walker A motif mediate the reversible transfer of a phosphoryl group between two adenosine diphosphate molecules to synthesize adenosine triphosphate and adenosine monophosphate. This activity, although rudimentary, bears resemblance to that of adenylate kinase (a P-loop NTPase enzyme). The polypeptides, dubbed as "P-loop prototypes", thus relate to contemporary P-loop NTPases in terms of their sequence and function, and yet, given their simplicity, serve as plausible representatives of the early "founder enzymes" involved in proto-metabolic pathways.

Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes.

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 αßα 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 αßα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

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.

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains.

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 spectroscopy (EPR) 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 revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding 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.

Bridging Themes: Short Protein Segments Found in Different Architectures.

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 20-80 residues that are unexpectedly shared between domains considered to have emerged independently. Among these "bridging themes" are ones shared between the most ancient domains, for example, 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.

Local fitness landscape of the green fluorescent protein.

Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

Enzyme Evolution: An Epistatic Ratchet versus a Smooth Reversible Transition.

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.

Quinone Methide-Based Organophosphate Hydrolases Inhibitors: Trans Proximity Labelers versus Cis Labeling Activity-Based Probes.

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.

Determining the interaction status and evolutionary fate of duplicated homomeric 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.