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Results for “ancestral sequence reconstruction”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes’ innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Science & Technology - Other Topics↗
DOE OSTI2020

Ancient origins of allosteric activation in a Ser-Thr kinase

A myriad of cellular events are regulated by allostery; therefore, evolution of this process is of fundamental interest. Here, we use ancestral sequence reconstruction to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2 (targeting protein for Xklp2), to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and has remained stable over 1 billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved, likely owing to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.

Science & Technology - Other Topics↗
DOE OSTI2020

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity

Abstract As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates—namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose–methanol–choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases—we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Evolution of Far-Red Light-Acclimated Photosystem II

Cyanobacteria are major contributors to global carbon fixation and primarily use visible light (400−700 nm) to drive oxygenic photosynthesis. When shifted into environments where visible light is attenuated, a small, but highly diverse and widespread number of cyanobacteria can express modified pigments and paralogous versions of photosystem subunits and phycobiliproteins that confer far-red light (FRL) absorbance (700−800 nm), a process termed far-red light photoacclimation, or FaRLiP. During FaRLiP, alternate photosystem II (PSII) subunits enable the complex to bind chlorophylls d and f, which absorb at lower energy than chlorophyll a but still support water oxidation. How the FaRLiP response arose remains poorly studied. Here, we report ancestral sequence reconstruction and structure-based molecular evolutionary studies of the FRL-specific subunits of FRL-PSII. We show that the duplications leading to the origin of two PsbA (D1) paralogs required to make chlorophyll f and to bind chlorophyll d in water-splitting FRL-PSII are likely the first to have occurred prior to the diversification of extant cyanobacteria. These duplications were followed by those leading to alternative PsbC (CP43) and PsbD (D2) subunits, occurring early during the diversification of cyanobacteria, and culminating with those leading to PsbB (CP47) and PsbH paralogs coincident with the radiation of the major groups. We show that the origin of FRL-PSII required the accumulation of a relatively small number of amino acid changes and that the ancestral FRL-PSII likely contained a chlorophyll d molecule in the electron transfer chain, two chlorophyll f molecules in the antenna subunits at equivalent positions, and three chlorophyll a molecules whose site energies were altered. The results suggest a minimal model for engineering far-red light absorbance into plant PSII for biotechnological applications.

ancestral sequence reconstruction↗
DOE OSTI2024

Substitution Models of Protein Evolution with Selection on Enzymatic Activity

Abstract Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme–substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.

Ferreiro, David↗
DOE OSTI2023

Molecular diversity and evolution of far-red light-acclimated photosystem I

The need to acclimate to different environmental conditions is central to the evolution of cyanobacteria. Far-red light (FRL) photoacclimation, or FaRLiP, is an acclimation mechanism that enables certain cyanobacteria to use FRL to drive photosynthesis. During this process, a well-defined gene cluster is upregulated, resulting in changes to the photosystems that allow them to absorb FRL to perform photochemistry. Because FaRLiP is widespread, and because it exemplifies cyanobacterial adaptation mechanisms in nature, it is of interest to understand its molecular evolution. Here, we performed a phylogenetic analysis of the photosystem I subunits encoded in the FaRLiP gene cluster and analyzed the available structural data to predict ancestral characteristics of FRL-absorbing photosystem I. The analysis suggests that FRL-specific photosystem I subunits arose relatively late during the evolution of cyanobacteria when compared with some of the FRL-specific subunits of photosystem II, and that the order Nodosilineales, which include strains like Halomicronema hongdechloris and Synechococcus sp. PCC 7335, could have obtained FaRLiP via horizontal gene transfer. We show that the ancestral form of FRL-absorbing photosystem I contained three chlorophyll f-binding sites in the PsaB2 subunit, and a rotated chlorophyll a molecule in the A0B site of the electron transfer chain. Along with our previous study of photosystem II expressed during FaRLiP, these studies describe the molecular evolution of the photosystem complexes encoded by the FaRLiP gene cluster.

ancestral sequence reconstruction↗
DOE OSTI2026

Exploring the binding properties and activities of ancestral expansins

Bacterial expansins are non-lytic proteins capable of loosening cellulose networks, offering promising applications in agriculture, biotechnology, and material science. Their ability to disrupt noncovalent interactions in biopolymer matrices such as cellulose and chitin positions them as valuable tools for upgrading abundant natural materials. However, their industrial use remains limited due to their relatively low wall-loosening activity compared to plant expansins. To address this limitation, we applied Ancestral Sequence Resurrection (ASR) to reconstruct and characterize ancient variants of the Bacillus subtilis expansin BsEXLX1. ASR is a powerful evolutionary tool that enables the inference and synthesis of ancestral proteins, allowing researchers to explore functional traits that may have been lost over time. This approach not only provides insights into protein evolution but also facilitates the design of proteins with enhanced properties, such as improved substrate affinity or structural stability. In this study, we combined biochemical and biophysical assays to evaluate the activity and binding behavior of ancestral expansins. Our results reveal that ancestral variants exhibit increased cellulose affinity, reduced binding to acidic polysaccharides, and greater salt resistance. Furthermore, these traits enhance their wall-loosening activity and demonstrate the utility of ASR in engineering surface-active proteins for industrial applications, particularly in biomass processing and cellulose modification.

09 BIOMASS FUELS↗
NASA NTRS2003

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65 degrees C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria, the distribution of thermophily in derived bacterial lineages, the inferred G + C content of ancient ribosomal RNA, and the geological record combined with assumptions concerning molecular clocks. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Bacteria/classification/metabolism↗
DOE OSTI2022

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of ‘common cause’ attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1998

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Non-NASA Center↗
NASA NTRS1998

Reconstructing Ancient Forms of Life

Progress in the past three months has occurred in two areas, reconstruction of ancestral proteins and improved understanding of chemical features that are likely to be universal in generic matter regardless of its genesis. Ancestral ribonucleases have been reconstructed, and an example has been developed that shows how physiological function can be assigned to in vitro behaviors observed in biological systems. Sequence data have been collected to permit the reconstruction of src homology 2 domains that underwent radiative divergence at the time of the radiative divergence of chordates. New studies have been completed that show how genetic matter (or its remnants) might be detected on Mars (or other non-terrean locations.) Last, the first in vitro selection experiments have been completed using a nucleoside library carrying positively charged functionality, illustrating the importance of non-standard nucleotides to those attempting to obtain evidence for an "RNA world" as an early episode of life on earth.

Benner, Steven A.↗
DOE OSTI2024

ContScout: sensitive detection and removal of contamination from annotated genomes

Contamination of genomes is an increasingly recognized problem affecting several downstream applications, from comparative evolutionary genomics to metagenomics. Here we introduce ContScout, a precise tool for eliminating foreign sequences from annotated genomes. It achieves high specificity and sensitivity on synthetic benchmark data even when the contaminant is a closely related species, outperforms competing tools, and can distinguish horizontal gene transfer from contamination. A screen of 844 eukaryotic genomes for contamination identified bacteria as the most common source, followed by fungi and plants. Furthermore, we show that contaminants in ancestral genome reconstructions lead to erroneous early origins of genes and inflate gene loss rates, leading to a false notion of complex ancestral genomes. Taken together, we offer here a tool for sensitive removal of foreign proteins, identify and remove contaminants from diverse eukaryotic genomes and evaluate their impact on phylogenomic analyses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An integrative framework reveals widespread gene flow during the early radiation of oaks and relatives in Quercoideae (Fagaceae)

ABSTRACT Although the frequency of ancient hybridization across the Tree of Life is greater than previously thought, little work has been devoted to uncovering the extent, timeline, and geographic and ecological context of ancient hybridization. Using an expansive new dataset of nuclear and chloroplast DNA sequences, we conducted a multifaceted phylogenomic investigation to identify ancient reticulation in the early evolution of oaks (Quercus). We document extensive nuclear gene tree and cytonuclear discordance among major lineages ofQuercusand relatives in Quercoideae. Our analyses recovered clear signatures of gene flow against a backdrop of rampant incomplete lineage sorting, with gene flow most prevalent among major lineages ofQuercusand relatives in Quercoideae during their initial radiation, dated to the Early‐Middle Eocene. Ancestral reconstructions including fossils suggest ancestors ofCastanea + Castanopsis,Lithocarpus, and the Old World oak clade probably co‐occurred in North America and Eurasia, while the ancestors ofChrysolepis, Notholithocarpus, and the New World oak clade co‐occurred in North America, offering ample opportunity for hybridization in each region. Our study shows that hybridization—perhaps in the form of ancient syngameons like those seen today—has been a common and important process throughout the evolutionary history of oaks and their relatives. Concomitantly, this study provides a methodological framework for detecting ancient hybridization in other groups.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1979

Molecular phylogenetic trees - On the validity of the Goodman-Moore augmentation algorithm

A response is made to the reply of Nei and Tateno (1979) to the letter of Holmquist (1978) supporting the validity of the augmentation algorithm of Moore (1977) in reconstructions of nucleotide substitutions by means of the maximum parsimony principle. It is argued that the overestimation of the augmented numbers of nucleotide substitutions (augmented distances) found by Tateno and Nei (1978) is due to an unrepresentative data sample and that it is only necessary that evolution be stochastically uniform in different regions of the phylogenetic network for the augmentation method to be useful. The importance of the average value of the true distance over all links is explained, and the relative variances of the true and augmented distances are calculated to be almost identical. The effects of topological changes in the phylogenetic tree on the augmented distance and the question of the correctness of ancestral sequences inferred by the method of parsimony are also clarified.

Holmquist, R.↗
NASA NTRS2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

nitrogen fixation↗
DOE OSTI2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

59 BASIC BIOLOGICAL SCIENCES↗