The origin of life required the emergence of metabolism, an autocatalytic network of enzymatic reactions that synthesize amino acids, nucleotides, and cofactors. At the origin of metabolism, there were no enzymes-how did it start? Empirical studies addressing early metabolic evolution are lacking. Harnessing protein structures for metabolic enzymes, we identify intermediate states in primordial metabolic assembly. We show that enzymatic metabolism in the universal common ancestor was incomplete, undergoing final assembly independently in the lineages leading to bacteria and archaea. Native transition metals-iron, cobalt, nickel, and palladium-served as the catalytic forerunners of both enzymes and cofactors at metabolic origin, while phosphite supplied energy, as it phosphorylates adenosine 5'-monophosphate to adenosine 5'-diphosphate and serine to phosphoserine using native metal catalysts in water. Phosphite and native metals occur in serpentinizing hydrothermal systems, identifying an energy-supplying, catalytic site of metabolic origin. Cofactors liberated nascent metabolism from native metal catalysts, engendering its autocatalytic state.

Recently in these pages, a paper by Lynch appeared in response to a report showing that his numbers for biosynthetic costs (ATP demand) in cells are inflated, so much so that they would require E. coli to obtain >100 ATP per glucose and mitochondria to obtain >240 ATP per glucose. The inflated estimates trace to one factor: Lynch exclusively considers ATP demand and systematically neglects ATP supply—the essence of bioenergetics. Thermodynamics stipulate that a cell cannot grow if its ATP demands exceed its ATP supply. Here I compare Lynch's calculated ATP demands to laboratory measurements of the ATP supply that E. coli synthesizes during cell division. The results bear out my case, and leave no doubt: Lynch's calculations require E. coli to synthesize ∼120 ATP per glucose, which is thermodynamically impossible. As a consequence, his demand-only ‘energetic’ attacks on mitochondria and endosymbiosis in evolution are baseless.

Apolipoprotein E (ApoE) is the primary cholesterol and lipid transporting apolipoprotein in the central nervous system (CNS) and is the greatest genetic risk factor for Alzheimer’s Disease (AD). There are three main isoforms differing by single amino acid changes: ε3 is “neutral”, ε4 is “risk” (Cys112Arg), and ε2 is “resilience” (Arg158Cys). Rare forms (Christchurch, Jacksonville) have also been proposed as resilience alleles, while an ε4-like allele (with Arg61Thr) is present in non-human primates without AD risk. To date, only a single mutated NMR structure of full-length ε3 in a closed conformation exists, leaving unanswered questions regarding conformational differences between isoforms. Additionally, nearly all experimental data for ApoE considers multimeric structures as opposed to the biologically relevant monomer. Here, we have integrated a graph representation of long-time scale molecular simulation data with neural networks to generate reversible Markov state models. Our method utilizes multiple replicates of long-timescale (six replicates of 15 µs per isoform, 540µs in total) to generate 200 starting conformations per isoform, each of which was simulated in triplicate for and additional 1 microsecond (600 µs per isoform). In total, 4.14 milliseconds of simulation across 6 isoforms were generated. We use Markov state models generated using GraphVAMPNets (a deep neural network utilizing graphs as the input data) in conjunction with DeepMSM, elucidating key macrostates for each isoform which may dictate the differences in conformation, informing the risk versus resilience potential. Finally, we integrated CoVAMPNet, a framework for directly comparing macrostates from different datasets. We have identified conformational differences between comparable macrostates which may inform differences in risk or resilience across the isoforms. This elucidation is a result of our integration of protein energy networks with molecular modeling, and may inform the development of future therapeutics targeting ApoE in AD. Mapping of macrostates to each other and comparing mean first passage times indicates potential risk and resilience states, with ε4 having lower transition times into a specific state and ε2 having a particularly stable state. We have identified putative risk or resilience conformational states in APOE, which may have implications for therapeutic discovery.

Energy conservation is crucial to life's origin and evolution. The common ancestor of all cells used ATP synthase to convert proton gradients into ATP. However, pumps generating proton gradients and lipids maintaining proton gradients are not universally conserved across all lineages. A solution to this paradox is that ancestral ATP synthase could harness naturally formed geochemical ion gradients with simpler environmentally provided precursors preceding both proton pumps and biogenic membranes. This runs counter to traditional views that phospholipid bilayers are required to maintain proton gradients. Here, we show that fatty acid membranes can maintain sufficient proton gradients to synthesize ATP by ATP synthase under the steep pH and temperature gradients observed in hydrothermal vent systems. These findings shed substantial light on early membrane bioenergetics, uncovering a functional intermediate in the evolution of chemiosmotic ATP synthesis during protocellular stages postdating the ATP synthase's origin but preceding the advent of enzymatically synthesized cell membranes.

Gene loss is an important process in genome evolution, though its power is often underestimated. If a gene is present at the root of a phylogenetic tree and can be lost in one lineage across the tree, it can potentially be lost in all, leading to gene extinction. Just before gene extinction, there will be one lineage that still retains the gene, generating a "last-one-out" distribution. Such an isolated gene presence will emulate the result of recent lateral gene acquisition, even though its distribution was generated by loss. How probable is it to observe "last-one-out" distributions in real data? Here, we mathematically derive this probability and find that it is surprisingly high, depending upon the tree and the gene loss rate. Examples from real data show that loss can readily account for observed frequencies of last-one-out gene distributions that might otherwise be attributed to lateral gene transfer.

Generic drugs have saved consumers billions of dollars in the United States. The demand for lower-cost and effective drugs, particularly for well-known peptide drugs like Ozempic and Wegovy (brand names for semaglutide), has resulted in a surge of generic drug development to address perceived shortages in the supply of the reference listed drugs (RLD). To address this demand for generics and expedite consumer access to lower-cost generic versions of approved drugs, the U.S. Food and Drug Administration (FDA) has developed an "Abbreviated New Drug Application" (ANDA) pathway that simplifies the generic drug review process and expands access to these much-needed medicines without compromising quality and safety standards. Guidelines for this pathway require sponsors to identify and characterize both process- and product-related impurities in drug formulations that differ in nature or concentration from the RLD. The ANDA pathway devotes specific attention to immunogenicity and recommends the use of orthogonal methods of assessment to demonstrate that a proposed generic drug is immunologically equivalent to its RLD and therefore suitable for submission via the ANDA pathway. In this perspective, we describe several orthogonal methods for immunogenicity risk assessment of generic peptide impurities and contrast these with other methods such as MHC-Associated Peptide Proteomics peptide elution (MAPPs) assays. Given their importance in the generic drug approval pathway, we have submitted the "PANDA®" immunogenicity risk assessment methods as a 'model master file'.

Studies by microbiologists in the 1970s provided robust estimates for the energy supply and demand of a prokaryotic cell. The amount of ATP needed to support growth was calculated from the chemical composition of the cell and known enzymatic pathways that synthesize its constituents from known substrates in culture. Starting in 2015, geneticists and evolutionary biologists began investigating the bioenergetic role of mitochondria at eukaryote origin and energy in metazoan evolution using their own, widely trusted-but hitherto unvetted-model for the costs of growth in terms of ATP per cell. The more recent model contains, however, a severe and previously unrecognized error that systematically overestimates the ATP cost of amino acid synthesis up to 200-fold. The error applies to all organisms studied by such models and leads to conspicuously false inferences, for example that the synthesis of an average amino acid in humans requires 30 ATP, which no biochemistry textbook will confirm. Their ATP 'cost' calculations would require that E. coli obtains ~100 ATP per glucose and that mammals obtain ~240 ATP per glucose, untenable propositions that invalidate and void all evolutionary inferences so based. By contrast, established methods for estimating the ATP cost of microbial growth show that the first mitochondrial endosymbionts could have easily doubled the host's available ATP pool, provided (i) that genes for growth on environmental amino acids were transferred from the mitochondrial symbiont to the archaeal host, and (ii) that the host for mitochondrial origin was an autotroph using the acetyl-CoA pathway. Stated in simple terms, the significance of these findings are this: Life is a chemical reaction. It requires energy release in order to proceed. The currency of energy in cells is adenosine triphosphate, ATP. Five decades ago, microbiologists were able to measure and understand the amount of ATP that cells require to grow. New studies by evolutionary biologists have appeared in the meantime that brush aside the older microbiological findings, using their own methods to calculate the ATP cost of growth instead. Science is, however, an imperfect undertaking. The new studies contain a major error, similar to conflating centimeters with yards. The error affects many publications and their conclusions. Using the old methods, we can still meaningfully study the role of energy in evolution, including the origin of complex, nucleus-bearing cells.

The history of Earth's atmospheric oxygen is a cornerstone of evolutionary biology. While unequivocal evidence for an increase in atmospheric O2 marks the Great Oxidation Event (GOE) roughly 2.4 billion years ago, evidence underlying proposals for pre-GOE O2 accumulation is debated. Here we have investigated the distribution of genes for oxygen reductases, the enzymes that consume O2 in respiratory chains, across independently generated molecular timescales of prokaryotic evolution. The data indicate that cytochrome bd-oxidases, heme-copper oxidases and alternative oxidases arose in the wake of the GOE ca. 2.4 billion years ago, after which the genes were subjected to abundant lateral gene transfer, a reflection of their utility in redox balance and membrane bioenergetics. The data lead us to propose a straightforward four-stage model for O2 accumulation surrounding the GOE: (i) Negligible O2 existed prior to the GOE. (ii) Cyanobacterial O2 production started at the GOE, yet was capped at 2 % [v/v] atmospheric O2, the threshold at which cyanobacterial nitrogenase is inhibited by O2. (iii) Production of 0.02 atm of O2 (2 % [v/v]) at the GOE buried roughly the entire atmospheric CO2 inventory, causing sudden enrichment of 13C in dissolved inorganic carbon (the Lomagundi 13C anomaly), through RuBisCO isotope discrimination, without atmospheric O2 exceeding 2 % [v/v]. (iv) High atmospheric 12C at the end of the Lomagundi excursion marks the origin of oxygen reductases, their rapid spread via function in respiratory CO2 liberation, and the onset of equilibrium between photosynthetic O2 production and respiratory O2 consumption at 2 % atmospheric O2.

Theories addressing the origin of life strive to explain the transition from rocks, water, inorganic carbon, and chondritic carbon (carbon from space) on the early Earth to living things (cells). The origin of life is generally regarded as the greatest of all evolutionary transitions and as one of the most difficult questions in the natural sciences. Energy, molecular organization, and the nature of the earliest life forms help to constrain the problem and link it to geochemical processes on the early Earth.

Serpentinizing hydrothermal vents are likely sites for the origin of metabolism because they produce H2 as a source of electrons for CO2 reduction while depositing zero-valent iron, cobalt, and nickel as catalysts for organic reactions. Recent work has shown that solid-state nickel can catalyze the H2-dependent reduction of CO2 to various organic acids and their reductive amination with H2 and NH3 to biological amino acids under the conditions of H2-producing hydrothermal vents and that amino acid synthesis from NH3, H2, and 2-oxoacids is facile in the presence of Ni0. Such reactions suggest a metallic origin of metabolism during early biochemical evolution because single metals replace the function of over 130 enzymatic reactions at the core of metabolism in microbes that use the acetyl-CoA pathway of CO2 fixation. Yet solid-state catalysts tether primordial amino synthesis to a mineral surface. Many studies have shown that pyridoxal catalyzes transamination reactions without enzymes. Here we show that pyridoxamine, the NH2-transferring intermediate in pyridoxal-dependent transamination reactions, is generated from pyridoxal by reaction with NH3 (as little as 5 mm) and H2 (5 bar) on Ni0 as catalyst at pH 11 and 80 degrees C within hours. These conditions correspond to those in hydrothermal vents undergoing active serpentinization. The results indicate that at the origin of metabolism, pyridoxamine provided a soluble, organic amino donor for aqueous amino acid synthesis, mediating an evolutionary transition from NH3-dependent amino acid synthesis on inorganic surfaces to pyridoxamine-dependent organic reactions in the aqueous phase.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

Corrinoids are cobalt-containing tetrapyrroles. They include adenosylcobalamin (vitamin B12) and cobamides that function as cofactors and coenzymes for methyl transfer, radical-dependent and redox reactions. Though cobamides are the most complex cofactors in nature, they are essential in the acetyl-CoA pathway, thought to be the most ancient CO2-fixation pathway, where they perform a pterin-to-cobalt-to-nickel methyl transfer reaction catalyzed by the corrinoid iron-sulphur protein (CoFeS). CoFeS occurs in H2-dependent archaeal methanogens, the oldest microbial lineage by measure of physiology and carbon isotope data, dating corrinoids to ca. 3.5 billion years. However, CoFeS and cobamides are also essential in the acetyl-CoA pathway of H2-dependent bacterial acetogens. To determine whether corrin biosynthesis was established before archaea and bacteria diverged, whether the pathways arose independently or whether cobamide biosynthesis was transferred from the archaeal to the bacterial lineage (or vice versa) during evolution, we investigated phylogenies and structural data for 26 enzymes of corrin ring and lower ligand biosynthesis. The data trace cobamide synthesis to the common ancestor of bacteria and archaea, placing it in the last universal common ancestor of all lifeforms (LUCA), while pterin-dependent methyl synthesis pathways likely arose independently post-LUCA in the lineages leading to bacteria and archaea. Enzymes of corrin biosynthesis were recruited from preexisting ancient pathways. Evolutionary forerunners of CoFeS function were likely Fe-, Ni- and Co-containing solid-state surfaces, which, in the laboratory, catalyze the reactions of the acetyl-CoA pathway from CO2 to pyruvate under serpentinizing hydrothermal conditions. The data suggest that enzymatic corrin biosynthesis replaced insoluble solid-state catalysts that tethered primordial CO2 assimilation to the Earth's crust, suggesting a role for corrin synthesis in the origin of free-living cells.

Life is an exergonic chemical reaction. The same was true when the very first cells emerged at life's origin. In order to live, all cells need a source of carbon, energy, and electrons to drive their overall reaction network (metabolism). In most cells, these are separate pathways. There is only one biochemical pathway that serves all three needs simultaneously: the acetyl-CoA pathway of CO2 fixation. In the acetyl-CoA pathway, electrons from H2 reduce CO2 to pyruvate for carbon supply, while methane or acetate synthesis are coupled to energy conservation as ATP. This simplicity and thermodynamic favorability prompted Georg Fuchs and Erhard Stupperich to propose in 1985 that the acetyl-CoA pathway might mark the origin of metabolism, at the same time that Steve Ragsdale and Harland Wood were uncovering catalytic roles for Fe, Co, and Ni in the enzymes of the pathway. Subsequent work has provided strong support for those proposals.In the presence of Fe, Co, and Ni in their native metallic state as catalysts, aqueous H2 and CO2 react specifically to formate, acetate, methane, and pyruvate overnight at 100 °C. These metals (and their alloys) thus replace the function of over 120 enzymes required for the conversion of H2 and CO2 to pyruvate via the pathway and its cofactors, an unprecedented set of findings in the study of biochemical evolution. The reactions require alkaline conditions, which promote hydrogen oxidation by proton removal and are naturally generated in serpentinizing (H2-producing) hydrothermal vents. Serpentinizing hydrothermal vents furthermore produce natural deposits of native Fe, Co, Ni, and their alloys. These are precisely the metals that reduce CO2 with H2 in the laboratory; they are also the metals found at the active sites of enzymes in the acetyl-CoA pathway. Iron, cobalt and nickel are relicts of the environments in which metabolism arose, environments that still harbor ancient methane- and acetate-producing autotrophs today. This convergence indicates bedrock-level antiquity for the acetyl-CoA pathway. In acetogens and methanogens growing on H2 as reductant, the acetyl-CoA pathway requires flavin-based electron bifurcation as a source of reduced ferredoxin (a 4Fe4S cluster-containing protein) in order to function. Recent findings show that H2 can reduce the 4Fe4S clusters of ferredoxin in the presence of native iron, uncovering an evolutionary precursor of flavin-based electron bifurcation and suggesting an origin of FeS-dependent electron transfer in proteins. Traditionally discussed as catalysts in early evolution, the most common function of FeS clusters in metabolism is one-electron transfer, also in radical SAM enzymes, a large and ancient enzyme family. The cofactors and active sites in enzymes of the acetyl-CoA pathway uncover chemical antiquity in metabolism involving metals, methyl groups, methyl transfer reactions, cobamides, pterins, GTP, S-adenosylmethionine, radical SAM enzymes, and carbon-metal bonds. The reaction sequence from H2 and CO2 to pyruvate on naturally deposited native metals is maximally simple. It requires neither nitrogen, sulfur, phosphorus, RNA, ion gradients, nor light. Solid-state metal catalysts tether the origin of metabolism to a H2-producing, serpentinizing hydrothermal vent.