Enigmatic evolution of microbial nitrogen fixation: insights from Earth’s past

Trends in Microbiology, Journal Year: 2023, Volume and Issue: 32(6), P. 554 - 564

Published: April 13, 2023

The evolution of nitrogen fixation undoubtedly altered nearly all corners the biosphere, given essential role in synthesis biomass. To date, there is no unified view on what planetary conditions gave rise to or how these have sustained it evolutionarily. Intriguingly, concentrations metals that nitrogenases require function changed throughout Earth's history. In this review, we describe interconnection metal and cycles with nitrogenase importance ancient ecology formation modern cycle. We argue exploration cycle's deep past will provide insights into humanity's immediate environmental challenges centered availability.

Language: Английский

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Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation

Molecular Biology and Evolution, Journal Year: 2024, Volume and Issue: 41(4)

Published: March 25, 2024

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations early evolved enzymes are unknown. Here, we bring one such mystery to relief by probing birth evolution G-subunit protein, an integral component certain members nitrogenase family, only capable biological nitrogen fixation. The is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after origin nitrogenases. We show arose with novel metal dependence expansion nitrogen-fixing microbes following transition in environmental availabilities atmospheric oxygenation began ∼2.5 ago. identify features suggest proteins mediated cofactor or interactions required for dependency, priming ancient nitrogenases their hosts exploit these newly diversified geochemical environments. further examined degree functional specialization extant ancestral homologs using laboratory reconstruction experiments. Our results indicate permanent recruitment depended prior establishment conserved showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

Language: Английский

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Reconstructing Early Microbial Life

Annual Review of Microbiology, Journal Year: 2024, Volume and Issue: 78(1), P. 463 - 492

Published: Aug. 21, 2024

For more than 3.5 billion years, life experienced dramatic environmental extremes on Earth. These include shifts from oxygen-less to overoxygenated atmospheres and cycling between hothouse conditions global glaciations. Meanwhile, an ecological revolution took place. Earth evolved one dominated by microbial containing the plants animals that are most familiar today. Many key cellular features early in history of life, collectively defining nature our biosphere underpinning human survival. Recent advances molecular biology bioinformatics have greatly improved understanding evolution across deep time. However, incorporation genetics, population biology, evolutionary approaches into study Precambrian biota remains a significant challenge. This review synthesizes current knowledge with emphasis ancient metabolisms. It also outlines foundations emerging interdisciplinary area integrates microbiology, paleobiology, synthetic reconstruct biological innovations.

Language: Английский

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Ancient nitrogenases are ATP dependent

mBio, Journal Year: 2024, Volume and Issue: 15(7)

Published: June 13, 2024

ABSTRACT Life depends on a conserved set of chemical energy currencies that are relics early biochemistry. One these is ATP, molecule that, when paired with divalent metal ion such as Mg 2+ , can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality extant biochemistry, it unclear whether ATP supported the function ancient enzymes. We investigate evolutionary necessity by experimentally reconstructing an ancestral variant N 2 -reducing enzyme nitrogenase. The Proterozoic ancestor predicted ~540–2,300 million years old, post-dating Great Oxidation Event. Growth rates under nitrogen-fixing conditions ~80% those wild type in Azotobacter vinelandii . In enzyme, hydrolysis two MgATP coupled electron transfer substrate reduction. has strict requirement for no other nucleotide triphosphate analogs (GTP, ITP, UTP) supporting activity. Alternative ions (Fe Co Mn ) activity but diminished activities compared similar enzyme. Additionally, shown identical efficiency per transferred two. Our results provide direct laboratory evidence usage IMPORTANCE energy-carrying molecules power many sustaining There may predate rise life Earth, how dependencies formed unknown. resurrection enzymes provides unique tool probe enzyme’s molecules, shedding light their biochemical origins. Through experimental reconstruction, this research investigates dependence carrier modern show resurrected does not have generalist specificity. Rather, like efficiency. findings elucidate early-evolved energy-yielding delineating role Ultimately, insights contribute unraveling intricate tapestry biology origins life-sustaining dependencies.

Language: Английский

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Understanding non-reducible N₂ in the mechanism of Mo-nitrogenase

Dalton Transactions, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

In my proposed mechanism of Mo-nitrogenase there are two roles for separate N2 molecules. One diffuses into the reaction zone between Fe2 and Fe6 where a strategic gallery H atoms can capture to form Fe-bound HNNH intermediate which is then progressively hydrogenated through intermediates containing HNNH2, NH NH2 entities NH3 in sequence. The second be parked an N2-pocket about 3.2 Å from or bind end-on at exo coordination site Fe2. This outside zone, not exposed atom donors, so 'non-reducible'. Here density functional calculations using 485+ model describe thermodynamics non-reducible moving exo-Fe2 position, resting state 19 mechanism. entropy component estimated included. result that all with ligation by NHx endo-Fe2 position free energy association negative. There remains some uncertainty status exo-Fe2-N2 during step H2 exchanges incoming reducible N2, least unbound molecules present. At it evident attainment octahedral stereochemistry dominates binding N2. Possibilities experimental support these computational conclusions discussed.

Language: Английский

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Nitrogen stable isotope fractionation by biological nitrogen fixation reveals cellular nitrogenase is diffusion limited

PNAS Nexus, Journal Year: 2025, Volume and Issue: 4(3)

Published: Feb. 25, 2025

Abstract Biological fixation of dinitrogen (N2), the primary natural source new bioavailable nitrogen (N) on Earth, is catalyzed by enzyme nitrogenase through a complex mechanism at its active site metal cofactor. How this reaction functions in cellular environments, including rate-limiting step, and how structure affects functioning remain unclear. Here, we investigated N2 N isotope effect (15εfix), measured as difference between 15N/14N ratios diazotroph net fixed substrate. The value 15εfix underpins cycle reconstructions differs diazotrophs using molybdenum-containing molybdenum-free nitrogenases. By examining for Azotobacter vinelandii strains with mutated nitrogenases, determined if reflects enzyme-scale effects and, thus, use efficiency. Distinct relatively stable values wild-type molybdenum- vanadium-nitrogenase isoforms (2.5‰ 5.8–6.6‰, respectively), despite changing growth rate electron availability, support proxy isoform type among extant Structural mutation access altered molybdenum-nitrogenase (3.0–6.8‰ α-70VI mutant). Structure-function isotopic modeling results indicated reduction rate-limited diffusion inside due to highly efficient catalysis cofactor, exemplifying tool probe mechanisms. Diffusion-constrained reactions could reflect structural tradeoffs that protect oxygen-sensitive cofactor from oxygen inactivation. This suggests function optimized modern oxygenated environments pre-Great Oxidative Event nitrogenases were less diffusion-limited potentially exhibited larger values.

Language: Английский

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Nitrogenase structural evolution across Earth’s history

Published: April 9, 2025

Life on Earth is more than 3.5 billion years old—nearly as old the age of planet. Over this vast expanse time, life and its biomolecules adapted to triggered profound changes Earth’s environment. Certain critical enzymes evolved early in history have persisted through planetary extremes. While sequence data widely used trace evolutionary trajectories, enzyme structure remains an underexplored resource for understanding how proteins evolve over long timescales. Here, we implement integrated approach study nitrogenase, ancient, globally essential nitrogen fixation. Despite ecological diversity host microbes, nitrogenase has strict functional limitations, including extreme oxygen sensitivity, energy requirements substrate availability. By combining phylogenetics, ancestral reconstruction, protein crystallography deep-learning based structural prediction, resurrected three history. We present first effort predict all extant structures along tree a total ∼5000 structures. Our lays foundation reconstructing key constraints that influence evolution studying ancient light phylogenetic environmental change.

Language: Английский

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Nitrogenase structural evolution across Earth’s history

Published: April 9, 2025

Life on Earth is more than 3.5 billion years old—nearly as old the age of planet. Over this vast expanse time, life and its biomolecules adapted to triggered profound changes Earth’s environment. Certain critical enzymes evolved early in history have persisted through planetary extremes. While sequence data widely used trace evolutionary trajectories, enzyme structure remains an underexplored resource for understanding how proteins evolve over long timescales. Here, we implement integrated approach study nitrogenase, ancient, globally essential nitrogen fixation. Despite ecological diversity host microbes, nitrogenase has strict functional limitations, including extreme oxygen sensitivity, energy requirements substrate availability. By combining phylogenetics, ancestral reconstruction, protein crystallography deep-learning based structural prediction, resurrected three history. We present first effort predict all extant structures along tree a total ∼5000 structures. Our lays foundation reconstructing key constraints that influence evolution studying ancient light phylogenetic environmental change.

Language: Английский

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Nitrogen fixation in the widely distributed marine γ-proteobacterial diazotroph Candidatus Thalassolituus haligoni

Science Advances, Journal Year: 2024, Volume and Issue: 10(31)

Published: July 31, 2024

The high diversity and global distribution of heterotrophic bacterial diazotrophs (HBDs) in the ocean has recently become apparent. However, understanding role these largely uncultured microorganisms play marine N

Language: Английский

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A CRISPR interference system for engineering biological nitrogen fixation

mSystems, Journal Year: 2024, Volume and Issue: 9(3)

Published: Feb. 20, 2024

ABSTRACT A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, globally consequential, nitrogenase-catalyzed reduction of atmospheric to bioavailable ammonia, vital area focus. Nitrogen fixation engineering relies upon extensive understanding underlying genetics microbial models, including broadly utilized gammaproteobacterium, Azotobacter vinelandii ( A. ). Here, we report first CRISPR interference (CRISPRi) system targeted gene silencing that integrates genomically via site-specific transposon insertion. We demonstrate CRISPRi can repress transcription an essential by ~60%. Further, show nitrogenase genes are suitably expressed from insertion site, indicating and engineered be co-integrated combinatorial studies expression engineering. Our established fills important gap desired purposes. IMPORTANCE All life on Earth requires survive. About 78% atmosphere alone nitrogen, yet humans cannot use it directly. Instead, obtain need our survival food eat. For more than 100 years, substantial portion agricultural productivity has relied industrial methods fertilizer synthesis, which consumes significant amounts nonrenewable energy resources exacerbates environmental degradation human-induced change. Promising alternatives these rely only biological pathway generating bioaccessible nitrogen: fixation. Bioengineering strategies require nitrogen-fixing microbes, but genetic tools this critical goal remain lacking. The report, developed bacterial model, , step toward elucidating complexity enabling their manipulation.

Language: Английский

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