The repertoire of protein architectures in proteomes is evolutionarily conserved and capable of preserving an accurate record of genomic history. Here we use a census of protein architecture in 185 genomes that have been fully sequenced to generate genome-based phylogenies that describe the evolution of the protein world at fold (F) and fold superfamily (FSF) levels. The patterns of representation of F and FSF architectures over evolutionary history suggest three epochs in the evolution of the protein world: (1) architectural diversification, where members of an architecturally rich ancestral community diversified their protein repertoire; (2) superkingdom specification, where superkingdoms Archaea, Bacteria, and Eukarya were specified; and (3) organismal diversification, where F and FSF specific to relatively small sets of organisms appeared as the result of diversification of organismal lineages. Functional annotation of FSF along these architectural chronologies revealed patterns of discovery of biological function. Most importantly, the analysis identified an early and extensive differential loss of architectures occurring primarily in Archaea that segregates the archaeal lineage from the ancient community of organisms and establishes the first organismal divide. Reconstruction of phylogenomic trees of proteomes reflects the timeline of architectural diversification in the emerging lineages. Thus, Archaea undertook a minimalist strategy using only a small subset of the full architectural repertoire and then crystallized into a diversified superkingdom late in evolution. Our analysis also suggests a communal ancestor to all life that was molecularly complex and adopted genomic strategies currently present in Eukarya.

Contemporary protein architectures can be regarded as molecular fossils, historical imprints that mark important milestones in the history of life. Whereas sequences change at a considerable pace, higher-order structures are constrained by the energetic landscape of protein folding, the exploration of sequence and structure space, and complex interactions mediated by the proteostasis and proteolytic machineries of the cell. The survey of architectures in the living world that was fuelled by recent structural genomic initiatives has been summarized in protein classification schemes, and the overall structure of fold space explored with novel bioinformatic approaches. However, metrics of general structural comparison have not yet unified architectural complexity using the 'shared and derived' tenet of evolutionary analysis. In contrast, a shift of focus from molecules to proteomes and a census of protein structure in fully sequenced genomes were able to uncover global evolutionary patterns in the structure of proteins. Timelines of discovery of architectures and functions unfolded episodes of specialization, reductive evolutionary tendencies of architectural repertoires in proteomes and the rise of modularity in the protein world. They revealed a biologically complex ancestral proteome and the early origin of the archaeal lineage. Studies also identified an origin of the protein world in enzymes of nucleotide metabolism harbouring the P-loop-containing triphosphate hydrolase fold and the explosive discovery of metabolic functions that recapitulated well-defined prebiotic shells and involved the recruitment of structures and functions. These observations have important implications for origins of modern biochemistry and diversification of life.

The standard molecular clock describes a constant rate of molecular evolution and provides a powerful framework for evolutionary timescales. Here, we describe the existence and implications of a molecular clock of folds, a universal recurrence in the discovery of new structures in the world of proteins. Using a phylogenomic structural census in hundreds of proteomes, we build phylogenies and time lines of domains at fold and fold superfamily levels of structural complexity. These time lines correlate approximately linearly with geological timescales and were here used to date two crucial events in life history, planet oxygenation and organism diversification. We first dissected the structures and functions of enzymes in simulated metabolic networks. The placement of anaerobic and aerobic enzymes in the time line revealed that aerobic metabolism emerged about 2.9 billion years (giga-annum; Ga) ago and expanded during a period of about 400 My, reaching what is known as the Great Oxidation Event. During this period, enzymes recruited old and new folds for oxygen-mediated enzymatic activities. Remarkably, the first fold lost by a superkingdom disappeared in Archaea 2.6 Ga ago, within the span of oxygen rise, suggesting that oxygen also triggered diversification of life. The implications of a molecular clock of folds are many and important for the neutral theory of molecular evolution and for understanding the growth and diversity of the protein world. The clock also extends the standard concept that was specific to molecules and their timescales and turns it into a universal timescale-generating tool.

Metabolism represents a complex collection of enzymatic reactions and transport processes that convert metabolites into molecules capable of supporting cellular life. Here we explore the origins and evolution of modern metabolism. Using phylogenomic information linked to the structure of metabolic enzymes, we sort out recruitment processes and discover that most enzymatic activities were associated with the nine most ancient and widely distributed protein fold architectures. An analysis of newly discovered functions showed enzymatic diversification occurred early, during the onset of the modern protein world. Most importantly, phylogenetic reconstruction exercises and other evidence suggest strongly that metabolism originated in enzymes with the P-loop hydrolase fold in nucleotide metabolism, probably in pathways linked to the purine metabolic subnetwork. Consequently, the first enzymatic takeover of an ancient biochemistry or prebiotic chemistry was related to the synthesis of nucleotides for the RNA world.enzyme activity ͉ evolution ͉ metabolism ͉ nucleotide metabolism T here is current interest in the processes underlying the biology of network because these offer insight into the organization and evolution of life (1). Cellular metabolism, one of the greatest achievements of science, is clearly the best-studied biological network. It represents a complex collection of enzymatic reactions and transport processes that convert metabolites into molecules capable of supporting cells and organisms. However, our knowledge of how modern metabolism originated and evolved is limited (2). One widely accepted hypothesis is that promiscuous catalytic activities in proteins provide a selective advantage and are recruited to perform new metabolic functions (3, 4). Considerable evidence supports a patchwork recruitment scenario in which recruited homologous enzymes are scattered over diverse pathways (2). For example, enzymes with ␣/␤ barrel fold structure that catalyze similar reactions occur across metabolic subnetworks (5, 6) and a small set of structural families dominates the small-molecule metabolism in Escherichia coli (7-10). The recruitment hypothesis assumes there is already an active enzymatic core with multifunctional enzymes from which proteins are drawn for metabolic innovation. Because history restricts the interplay between structure and function of metabolic enzymes, we here use evolutionary patterns in protein structure advantageously to study recruitment processes and metabolic network evolution.The protein world has a hierarchical and redundant organization specified in terms of evolutionary units of molecular structure, the protein domains (11). Domains are generally unified into a comparatively small set of folding architectures, protein superfamilies, and these are further grouped into protein folds (12). Domain structure is generally maintained for long periods of evolutionary time. Consequently, the discovery of an architectural design constitutes an important and rare event in evolutionary history....

The origin of life has puzzled molecular scientists for over half a century. Yet fundamental questions remain unanswered, including which came first, the metabolic machinery or the encoding nucleic acids. In this study we take a protein-centric view and explore the ancestral origins of proteins. Protein domain structures in proteomes are highly conserved and embody molecular functions and interactions that are needed for cellular and organismal processes. Here we use domain structure to study the evolution of molecular function in the protein world. Timelines describing the age and function of protein domains at fold, fold superfamily, and fold family levels of structural complexity were derived from a structural phylogenomic census in hundreds of fully sequenced genomes. These timelines unfold congruent hourglass patterns in rates of appearance of domain structures and functions, functional diversity, and hierarchical complexity, and revealed a gradual build up of protein repertoires associated with metabolism, translation and DNA, in that order. The most ancient domain architectures were hydrolase enzymes and the first translation domains had catalytic functions for the aminoacylation and the molecular switch-driven transport of RNA. Remarkably, the most ancient domains had metabolic roles, did not interact with RNA, and preceded the gradual build-up of translation. In fact, the first translation domains had also a metabolic origin and were only later followed by specialized translation machinery. Our results explain how the generation of structure in the protein world and the concurrent crystallization of translation and diversified cellular life created further opportunities for proteomic diversification.