Biology subjects

Kacar, B.

Publications and source records attributed to Kacar, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Evolution-guided engineering of an ancient nitrogenase interface enhances enzyme activity and stability

Nitrogenase is the only enzyme capable of biological nitrogen fixation and a major target for sustainable agricultural engineering, yet its functional and structural complexity has made it difficult to identify regions that can be modified without compromising activity or assembly. Here, we use structural evolution to identify a lineage-specific N-terminal extension in NifK as a candidate engineering target within the MoFe protein interface. By generating variant libraries exceeding 9,000 members and evaluating them through deep mutational scanning, diazotrophic growth assays and in vitro characterization to map the sequence-function landscape of this interface across variable conditions. We show that NifK extension is required for nitrogenase activity while remaining broadly tolerant to mutation, revealing a sequence-function landscape that is both flexible and constrained. A subset of residues at the NifD-NifK interface are critical for maintaining complex stability. Structural analyses indicate that the extension stabilizes the MoFe complex via co-evolved electrostatic interactions, and targeted mutations can improve both enzymatic activity and thermostability. These findings identify the NifK extension as a tunable interface, providing a strategy for engineering more robust nitrogenase variants.

evolutionary biology↗

bioRxiv2026

Paralogous guanine deaminases likely acquired from bacteria by horizontal gene transfer promote purine homeostasis in Caenorhabditis elegans

Disruptions in purine metabolism contribute to a range of human diseases, from rare genetic disorders such as Lesch-Nyhan syndrome and xanthinuria to common conditions including gout and cancer. To better understand the metabolic networks that regulate purine homeostasis, we developed a Caenorhabditis elegans model of xanthine dehydrogenase (xdh-1) deficiency. Remarkably, xdh-1 mutant animals form rare xanthine stones, recapitulating a hallmark of human xanthinuria. To uncover genetic regulators of purine homeostasis, we performed a forward genetic screen for mutations that exacerbate xanthine stone formation in xdh-1 mutants. This approach identified multiple loss-of-function alleles in a previously uncharacterized gene, which we named gda-1. We show that gda-1 encodes an intestinal guanine deaminase that mediates a key enzymatic step in purine catabolism. The C. elegans genome also encodes a paralog, gda-2, which shares guanine deaminase activity but is expressed in distinct tissues. While gda-2 can compensate for gda-1 loss in guanine metabolism, the two genes exhibit non-redundant roles in regulating xanthine accumulation and stone formation. Interestingly, our evolutionary analyses suggest that gda-2 was acquired by nematodes via horizontal gene transfer from bacteria. These findings reveal a spatially regulated purine catabolism pathway in C. elegans and suggest that acquisition of bacterial genes has shaped a core nematode metabolic network.

genetics↗

bioRxiv2026

Evolution of Translation Initiation Factor 2 Extensions Links Initiation to Bacterial Stress Response

Terminal extensions are recurrent features in protein evolution, often linked to environmental adaptation and novel regulatory or interaction functions. Here, we combine comparative genomics, structural modeling, and functional assays to elucidate the evolutionary diversification and functional significance of one of the key proteins of all cells, translation initiation factor 2 (IF2) terminal extensions across the tree of life. Specifically, we reconstruct the first comprehensive evolutionary map of IF2 across life, analyzing [~]800 homologs and classify seven distinct structural architectures of IF2 based on extension regions. These extensions are enriched for intrinsically disordered and phase-separation-promoting residues, suggesting roles beyond the conserved catalytic core. Further, functional characterization of IF2 with varying N-terminal lengths show that loss of the N-terminal extension slows bacterial growth specifically under temperature and pH stress. Appending C-terminal extensions from different organisms to the Escherichia coli IF2 demonstrates a conserved role for these extensions in adaptation to temperature and anaerobiosis. Our findings establish the functional significance of IF2 terminal extensions, linking their evolutionary diversification to stress-dependent regulation of translation.

microbiology↗

bioRxiv2025

Agnostic Tools for Life Detection: A Multiscale Approach to Biological and Chemical Reaction Networks

Life manifests itself across a variety of different operative scales, ranging from molecular networks up to ecologies. Deep conceptual difficulties arise when trying to define exactly what lifes distinguishing attributes are, and how they may be quantitatively distinguished from abiotic systems that are complicated but demonstrably not alive. Here we assembled different biotic and abiotic chemical reaction networks to test whether network-level (macroscale) and reaction-level (microscale) metrics can impartially distinguish biotic from abiotic category networks. Macroscale attributes such as statistical tests for a discrete networks heavy-tailed connectivity distribution generally distinguish bona fide biotic and protobiotic networks (E. coli heterotrophy, pruned protometabolism, KEGG) from abiotic networks but with two significant exceptions (radiolytic network and the Open Reaction Database). Microscale attributes such as an analysis of the frequency of a set of six primitive reaction motifs shows a depletion of some motifs in all chemical networks and the prevalence of others in specific networks; all networks were readily distinguishable from random network variants across all motifs. A comparison of the chemical spaces spanned by the different networks points to similarity and dissimilarity relationships between networks. Independent component analysis of an aggregate of all measured attributes, across all contexts, reliably distinguishes abiotic, prebiotic and biotic networks from one another. The combination of macro- and microscale attributes forms an agnostic toolset that may help to evaluate the prebiotic plausibility of different candidate settings for the origins of life, and may inform new ways of detecting and recognizing living systems (engineered or extraterrestrial) that differ from Terran biochemistry.

systems biology↗

bioRxiv2025

Microbial N2O reduction in sulfidic waters:Implications for Proterozoic oceans

Throughout Earths history, shifts in ocean redox influenced the bioavailability of trace metals, shaping the activity of microorganisms. In Proterozoic oceans, the precipitation of copper (Cu) with sulfide was hypothesized to limit the bioavailability of Cu. This limitation may have suppressed microbial reduction of nitrous oxide (N2O), due to the Cu dependency of nitrous oxide reductase (Nos). It is thought that without this critical microbial sink, Proterozoic oceans were a significant net source of N2O. Here, we revisit this paradigm in light of recently derived [~]20-fold lower estimates for sulfide in Proterozoic seawater and an empirical evaluation of the potential for microbial N2O reduction under sulfidic conditions. Leveraging publicly available environmental metatranscriptomes, we infer active N2O reduction from the detection of nosZ transcripts in multiple marine and lacustrine systems in which sulfide and Cu concentrations are analogous to those of the Proterozoic. In controlled culture experiments, we demonstrate that the purple non-sulfur bacterium Rhodopseudomonas palustris can reduce N2O at sulfide concentrations up to 100 {micro}M, well above levels predicted for Proterozoic oceans. Based on trace metal speciation modeling, we suggest that Cu remains bioavailable under Proterozoic-like conditions as a dissolved CuHS0 complex. Using phylogenetics, we infer that early N2O reducers were probably anoxygenic phototrophs and performed N2O reduction as dark metabolism. Collectively, these observations suggest microbial N2O reduction occurs under euxinic conditions, implying that Proterozoic marine N2O emissions were substantially lower than previously proposed. Our conclusions inform our understanding of the microbial ecology in sulfidic waters, the early climate, and the search for extraterrestrial life.

ecology↗

bioRxiv2025

ChemOrigins: A community curated database for origins of life chemistry

The origin of life is one of the most compelling questions in science. While experimental prebiotic chemistry has produced a wide range of reactions and plausible pathways, the resulting data remain fragmented across numerous publications and disciplinary journals. Here, we introduce ChemOrigins, an open-access, community-curated knowledge graph that organizes experimentally supported prebiotic reactions. By representing molecules, reactions, conditions, and literature sources as interconnected nodes, ChemOrigins enables modular grouping of reactions and supports complex, query-driven exploration via a graph database architecture. We demonstrate the utility of this framework through text-based searches, reaction network expansions, and the interactive visualization of user-annotated chemical modules. Unlike generative models, ChemOrigins prioritizes curated, evidence-based content and fosters community contributions through expert annotations and a user-friendly interface. As a structured resource, ChemOrigins is designed to complement existing chemical databases and serve as a foundation for computational, educational, and theoretical research in the origins-of-life field.

bioinformatics↗

bioRxiv2025

Did Iron Suppress Eukaryote Emergence and Early Radiation?

The last eukaryotic common ancestor (LECA) is widely thought to have been an oxygen-respiring organism, arising through endosymbiosis when a free-living bacterium became the mitochondrion. Owing to the mitochondrions central metabolic task of oxidative phosphorylation, oxygen availability has long been a hypothesized driver of eukaryogenesis. However, this hypothesis is challenged by a temporal disconnect, spanning several hundred million years, between the earliest geochemical evidence for oxygen in the environment ([~]3.2-2.5 Ga) and the oldest widely accepted eukaryotic fossils ([~]1.7 Ga). Notably, the earliest candidate eukaryotes appear contemporaneous with the cessation of major iron deposits and rise of sulfide- and sulfate-rich marine sediments in coastal environments. Here, we integrate Proterozoic surface geochemical records with the microbial biochemistry of iron to examine potential environmental constraints on early eukaryotic evolution. Iron bioavailability exerts complex and often antagonistic effects on both aerobic and anaerobic microbial lineages that contributed to LECA. Elevated iron levels likely disrupted cellular homeostasis, particularly by destabilizing labile iron pools and promoting oxidative damage to bacterial lipids. The programmed cell death pathways known as ferroptosis, which is widespread among eukaryotic lineages, may trace its origins to iron-rich conditions in Archaean and Paleoproterozoic seawater and LECA. Our findings challenge oxygen-centered paradigms of eukaryogenesis and reframes the long-recognized temporal gap as a consequence of iron-mediated physiological constraints.

evolutionary biology↗

bioRxiv2025

Persistent nitrogenase isotope signatures over two billion years of engineered evolution

Nitrogen isotope fractionation ({varepsilon}15N) in sedimentary rocks has provided evidence for biological nitrogen fixation, and thus primary productivity, on the early Earth. However, the extent to which molecular evolution has influenced the isotopic signatures of nitrogenase, the enzyme that catalyzes the conversion of atmospheric nitrogen (N2) to bioavailable ammonia, remains unresolved. Here, we reconstruct and experimentally characterize a library of synthetic ancestral nitrogenase genes, spanning over 2 billion years of evolutionary history. By engineering modern microbes to express these ancient nitrogenases, we assess the resulting {varepsilon}15N values under controlled laboratory conditions. All engineered strains exhibit {varepsilon}15N values within a narrow range comparable to that of modern microbes, suggesting that molybdenum (Mo)-dependent nitrogenase has been largely invariant throughout evolutionary time since the origins of this pathway. These results confirm the robustness of N-isotope biosignatures in the ancient rock record and bolster their utility in the search for life in extraterrestrial environments.

evolutionary biology↗

bioRxiv2025

Ecological constraints and evolutionary trade-offs shape nitrogen fixation across habitats

From its earliest beginnings, lifes expansion into new habitats has been profoundly shaped by its reciprocal interactions with Earths changing environments. Understanding how ancient metabolisms co-evolved with their environments requires uncovering the ecological and evolutionary processes that structured the functionally linked genes and networks underlying these metabolisms. Here, we focus on nitrogen (N2) fixation, one of lifes most critical metabolisms, and investigate the drivers of complexity in its associated gene machinery today. We used a large-scale comparative genomics framework to construct a comprehensive catalog of extant N2 fixation-associated genes and assessed their distribution across diverse microbial genomes and environmental backgrounds. Genomes enriched in N2 fixation genes generally have larger genome sizes, broader metabolic capabilities, wider habitat ranges, and are predominantly associated with mesophilic and aerobic lifestyles. Evolutionary reconstructions reveal a pattern of early gene gains in ancestral diazotrophs followed by lineage-specific gene losses in later diverging taxa, suggesting evolutionary trade-offs shaped by changing environments. These findings demonstrate that the evolution of N2 fixation has been intertwined with the composition and organization of the genes supporting the overarching N2 metabolism, driven by feedback between genome evolution and shifting environmental and ecological conditions.

microbiology↗

bioRxiv2025

Biological molybdenum usage stems back to 3.4 billion years ago

Molybdenum (Mo) is an essential nutrient for most living organisms, serving as a cofactor in a diverse array of molybdoenzymes that catalyze key reactions in several elemental cycles. However, geochemical data suggest that dissolved Mo concentrations in the Archean ocean (before 2.5 billion years ago) were 1-2 orders of magnitude lower than today, raising questions about its bioavailability to early life. Here, we apply a phylogenomic approach to chart the modern biological and environmental distribution of Mo-related enzymes and use phylogenetic reconciliations to reconstruct the evolutionary history of biological Mo usage. Our results reveal the ubiquity of molybdoenzymes across contemporary organisms inhabiting diverse environments. Furthermore, phylogenetic evidence indicates that the earliest molybdoenzymes stem back to the Paleo/Mesoarchean (~3.5-3.0 Gya), facilitating critical energy-harnessing reactions in some of Earths most ancient life forms. Taken together, our findings challenge the prevailing view of limited Mo bioavailability on the anoxic early Earth.

evolutionary biology↗

bioRxiv2024

Ecological resource competition as a driver of metallome evolution

The oldest nitrogenase isozyme, emerging a billion years or more before the Great Oxidation Event (GOE), required a molybdenum (Mo)-based cofactor. "Alternative" nitrogenases using iron (Fe) or vanadium (V) cofactors evolved only after the GOE. This history is puzzling because environmental Fe availability decreased after the GOE, while Mo availability increased, due to the contrasting environmental redox behaviors of these elements. Why, then, did the alternatives emerge only after the GOE? Using a model constrained by known microbial Mo quotas, we demonstrate that a strong selection pressure for the use of metals in nitrogenase other than Mo is a plausible consequence of competition between nitrogen-fixing prokaryotes and nitrate-reducing microbes, which require Mo for nitrate reduction and assimilation. This competition would have intensified after the GOE due to increasing availability of nitrate, explaining the evolutionary timing of nitrogenases isozymes. Ecological resource competition therefore emerges as a third driver of metallome evolution in deep-time, alongside the relative environmental availabilities and adaptive advantages of particular metals.

evolutionary biology↗

bioRxiv2024

Nitrogenase structural evolution across Earth s history

Life on Earth is about 4 billion years old--nearly as old as the planet itself. Over this immense timespan, living systems and their biomolecules have both adapted to and driven profound changes in the Earths environment. Among these, certain critical enzymes emerged early and have persisted through planetary extremes. Here, we implement an integrated approach to investigate the structural evolution of nitrogenase, an ancient and globally essential enzyme responsible for biological nitrogen fixation. Despite the ecological diversity of its host microbes, nitrogenase retains strict functional constraints, including extreme oxygen sensitivity, high energy demands, and substrate availability. We combined phylogenetics, ancestral sequence reconstruction, protein crystallography and deep-learning based structural prediction to resurrect nearly three billion years of nitrogenase structural history. This effort represents the first effort to predict the full set of extant and ancestral structures along the evolutionary tree of a single enzyme, yielding over 5000 structural models. Our framework lays a foundation for reconstructing key structural constraints that shape protein evolution and examining ancient enzymes within the broader context of phylogenetic relationships and environmental transitions across geological timescales.

evolutionary biology↗

bioRxiv2024

Molecular and structural innovations of the stator motor complex at the dawn of flagellar motility

The rotation of the bacterial flagellum is powered by the MotAB stator complex, which converts ion flux into torque. The origin and evolution of this remarkable complex is understudied. Here, we perform the first phylogenetic and structural characterisation and classification of MotAB and nonflagellar relatives. Using 193 genomes sampled across 27 bacterial phyla, we estimated phylogenies and ancestral sequences, and generated AlphaFold predictions for all extant and reconstructed proteins. We then mapped them onto the phylogeny to determine patterns of diversity and distribution of structural innovations. We identify two discrete groups: the Flagellar Ion Transporters (FIT) and the Generic Ion Transporters (GIT). The FIT proteins are structurally conserved and have a square fold domain and a torque-generating interface (TGI). FIT proteins are divided into two clades, termed TGI4 and TGI5, referring to whether there have 4 or 5 short helices in the TGI. TGI5 motors are predominantly found in Proteobacteria and include the well-studied E. coli K12 system, while TGI4 motors are found in diverse phyla and include the Na+-powered polar motors of Vibrio (PomAB). The GIT proteins, on the other hand, are structurally diverse and lack these attributes. The interaction between the A and B subunits is conserved across the FIT and GIT proteins. The two subunits are jointly necessary for function, with the genes typically adjacent within an operon. Motility assays in E. coli show that the structural elements unique to FIT play an important role in flagellar motility. Our results indicate that the stator motor complex has a single origin and shares unique motility-related structural traits. Significance StatementFlagellar motility is a key feature in bacterial pathogenicity and survival. It allows bacteria to propel themselves and direct movement according to environmental conditions. We investigated the molecular and structural diversity of the stator motor proteins that provide the ion motive force to power flagellar rotation. This study integrates phylogenetics, 3D protein structure modeling, motility assays and ancestral state reconstruction (ASR) to provide insights into the structural mechanisms that first powered the flagellar motor. We provide the first phylogenetic and structural characterisation and classification of MotAB and relatives.

evolutionary biology↗

bioRxiv2024

Ancestral structure prediction reveals the conformational impact of the RuBisCO small subunit across time

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an ancient protein critical for CO2-fixation and global biogeochemistry. Form-I RuBisCO complexes uniquely harbor small subunits that form a hexadecameric complex together with their large subunits. The small subunit protein is thought to have significantly contributed to RuBisCOs response to the atmospheric rise of O2 [~]2.5 billion years ago, marking a pivotal point in the enzymes evolutionary history. Here, we performed a comprehensive evolutionary analysis of extant and ancestral RuBisCO sequences and structures to explore the impact of the small subunits earliest integration on the molecular dynamics of the overall complex. Our simulations suggest that the small subunit restricted the conformational flexibility of the large subunit early in its history, impacting the evolutionary trajectory of the Form-I RuBisCO complex. Molecular dynamics investigations of CO2 and O2 gas distribution around predicted ancient RuBisCO complexes suggest that a proposed "CO2 reservoir" role for the small subunit is not conserved throughout the enzymes evolutionary history. The evolutionary and biophysical response of RuBisCO to changing atmospheric conditions on ancient Earth showcase multi-level and trackable responses of enzymes to environmental shifts over long timescales.

evolutionary biology↗

bioRxiv2024

The Origins of ATP Dependence in Biological Nitrogen Fixation

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg2+, can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the key N2-reducing enzyme nitrogenase. We show that the ancestor has a strict requirement for ATP and its hydrolysis is coupled to electron transfer for N2 reduction. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme, and underscore how biomolecular constraints can entirely decouple cofactor selection from environmental availability.

evolutionary biology↗

bioRxiv2023

A CRISPR interference system for the nitrogen-fixing bacterium Azotobacter vinelandii

A grand challenge for the next century can be found in mitigating the effects of changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a particularly vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by [~]60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system extends the utility of A. vinelandii and will aid efforts to engineer microbial nitrogen fixation for desired purposes. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/565814v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@128b75forg.highwire.dtl.DTLVardef@1ecdf4org.highwire.dtl.DTLVardef@6363c6org.highwire.dtl.DTLVardef@12b8358_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

A beneficial synonymous substitution in EF-Tu is contingent on genetic background

Synonymous mutations are changes to DNA sequence that occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation Elongation Factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels, as well as the polysome abundance on global transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes. ImportanceThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein Elongation Factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

evolutionary biology↗

bioRxiv2023

A hybrid nitrogenase with regulatory elasticity 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, engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs restrict the available 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 nitrogenase sequence-level perturbations. Rather, physiological compatibility with the ancestral nitrogenase variant is restored by reducing trace metal and electron resource allocation to nitrogenase. Our results spotlight cellular processes adjacent to nitrogen fixation as productive engineering targets to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCEAzotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and remodeled nitrogenase variants can be modulated at the regulatory level. Engineered cells respond by adjusting expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

microbiology↗