Biology subjects

Reif-Trauttmansdorff, T.

Publications and source records attributed to Reif-Trauttmansdorff, T..

8 recordsLinked to original sources

bioRxiv2026

Interface swapping orchestrates carbon transfer in the archaeal acetyl-CoA decarbonylase/synthase

The Wood-Ljungdahl pathway is one of biologys most ancient routes for carbon fixation and energy metabolism, used by organisms such as methanogenic archaea. One of its central metabolic complexes is the acetyl-CoA decarbonylase/synthase (ACDS) complex, catalyzing acetyl-CoA synthesis and cleavage through the co-ordinated action of carbon monoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS), and corrinoid iron-sulfur protein (CoFeSP). Unlike bacterial CODH/ACS, archaeal ACDS lacks a stable bifunctional CODH-ACS architecture, raising the question of how reactive CO and methyl intermediates are efficiently transferred between catalytic modules. Using cryo-electron microscopy, crosslinking mass spectrometry, small-angle X-ray scattering, and biophysical analyses, we resolved the organization and dynamics of the [~]2 MDa archaeal ACDS supercomplex from Methanosarcina acetivorans. We identified CoFeSP as a central architectural scaffold that self-assembles into hexa- to octameric oligomers via a conserved N-terminal region of the CdhD subunit. This scaffold likely tethers CODH and ACS through conserved disordered terminal regions, positioning the catalytic modules in the complexs periphery. We propose a mechanism in which ACS transiently alternates between CODH and CoFeSP, enabling efficient CO and methyl-group transfer without stable binary complexes. This dynamic organization represents a fundamental difference to the stable bifunctional CODH/ACS in bacteria, highlighting how transient interactions enable efficient acetyl-CoA metabolism in archaea.

biochemistry↗

bioRxiv2026

The anaerobic cryo-EM structure of the methanogenic Mtr complex reveals a nitrogenase-like cluster bound to its active site

Methanogenic archaea conserve energy by coupling methyl-group transfer to the generation of a chemiosmotic sodiumion (Na+) gradient. This central energy-conserving step is catalyzed by the membrane-bound N5-methyl-H4MPT:coenzyme M methyltransferase (Mtr). Here, we present high-resolution cryo-electron microscopy structures of the Mtr complex from Methanosarcina mazei determined under strictly anaerobic conditions. The structures reveal an unexpected, electron-dense metallocluster embedded within the central cavity of the MtrCDE trimer in the membrane plane. Based on the unique topology and density we modeled it as an [Fe8S9C] L-type cluster. It is positioned adjacent to both the coenzyme M substrate and the corrinoid cofactor of MtrA in the MtrA-MtrCDE engaged state, thereby being located right at the catalytic core of the enzyme. We could further show that binding of MtrA to MtrCDE triggers rearrangements within the interface of MtrDE that widen a putative ion-conduction pathway. The proximity of the conserved sodium-binding site to the catalytic center suggests a putative link between methyl-transfer chemistry and Na+ translocation. In a broader context, these findings improve our understanding of how methyl transfer, analogous to redox chemistry, can drive chemiosmotic energy conversion.

biochemistry↗

bioRxiv2026

Coupling of electron-bifurcation modules powers aromatic ring reduction beyond the biological redox window

Microbial degradation of ubiquitous aromatic compounds is central to the global carbon cycle and bioremediation, yet the intrinsic stability of aromatic rings poses a major barrier to their breakdown. Under strictly anaerobic conditions, class II benzoyl-CoA reductases (BCRII) catalyse the key step of this process, a Birch-like reduction of the aromatic ring to a cyclic diene at a tungsten cofactor. This reaction operates beyond the redox limits of conventional biological electron transfer, yet the mechanism by which BCRII generates such extreme reducing power has remained unclear. Here, high-resolution cryo-electron microscopy, in situ cryo-electron tomography, and enzymatic analyses reveal that the one-MDa BCRII complex from Geobacter metallireducens links two distinct flavin-based electron-bifurcation modules, previously characterised in hydrogenases and heterodisulfide reductases, to drive aromatic ring reduction. Reduced ferredoxin and NADH deliver electrons through sequential confurcation and bifurcation to the catalytic site, while cryo-electron tomography of native cells identifies electron-transferring flavoprotein as the second acceptor directing high-potential electrons into the respiratory chain. These results show that modular FBEB units can be hierarchically assembled to extend metabolic redox capacity, highlighting their versatility as adaptable components for electron transfer pathways.

biochemistry↗

bioRxiv2025

Structure of the energy converting methyltransferase (Mtr) of Methanosarcina mazei in complex with an oxygen-stress responsive small protein

Methanogenic archaea contribute 1-2 Gt of methane annually, impacting both global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr complex), which catalyzes the methyl transfer between two methanogen specific cofactors. This exergonic methyl transfer step is coupled with a vectorial sodium ion transport from the cytoplasm to the cell exterior and is the only energy conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 [A] single-particle cryo-EM structure of the full Mtr complex from Methanosarcina mazei. Our structural model encompasses the entire complex, reveals the arrangement of archaeal phospholipids, the architecture of the sodium ion binding site, and the structure and interactions of all catalytic subunits. Most strikingly, we discover and characterize MtrI, a previously unannotated small open reading frame (small ORF), encoded protein (<100 aa) conserved across the order of Methanosarcinales. MtrI binds to the cytoplasmic domain of MtrA in response to oxygen exposure, suggesting a role in oxygen stress response and protection. By binding on top of the sodium channel and anchoring to the cobamide cofactor in MtrAs cytoplasmic domain, MtrI might prevent sodium leakage and inhibit MtrA-CoM turnover. These findings offer new insights into methanogen energy conservation and uncover a potential adaptive response to oxygen exposure, expanding our understanding of methanogen survival strategies under oxidative stress.

biochemistry↗

bioRxiv2024

Methylthio-alkane reductases use nitrogenase metalloclusters for carbon-sulfur bond cleavage

Methylthio-alkane reductases convert methylated sulfur compounds to methanethiol and small hydrocarbons, a process with important environmental and biotechnological implications. These enzymes are classified as nitrogenase-like enzymes, despite lacking the ability to convert dinitrogen to ammonia, raising fundamental questions about the factors controlling their activity and specificity. Here, we present the first molecular structure of the methylthio-alkane reductase, which reveals large metalloclusters, including the P-cluster and the [Fe8S9C]-cluster, previously only found in nitrogenases. Our findings suggest that distinct metallocluster coordination, surroundings, and substrate channels, determine the activity of these related metalloenzymes. This study provides new insights into nitrogen fixation, sulfur-compound reduction, and hydrocarbon production. We also shed light on the evolutionary history of P-cluster and [Fe8S9C]-cluster-containing reductases emerging prior to nitrogenases.

biochemistry↗

bioRxiv2024

Molecular principles of redox-coupled sodium pumping of the ancient Rnf machinery

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD+ and pumps sodium ions (Na+) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na+-pumping NADH-quinone oxidoreductase (Nqr)1. By running in reverse, Rnf reduces ferredoxin with NADH as reductant at the expense of the transmembrane electrochemical ion gradient and provides low potential electrons for nitrogenases as well as CO2 reductases. Yet, the molecular principles that couple the long-range electron transfer to the Na+ translocation across the membrane remain elusive. Here we resolve key functional states along the electron transfer pathway using redox-controlled cryo-electron microscopy (cryo-EM) that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na+ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster in the vestibule between the RnfA/E subunits electrostatically attracts the sodium ions, and in turn, triggers an inward/outward transition with alternating membrane access driving the Na+ pump and the reduction of NAD+. Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.

biochemistry↗

bioRxiv2024

2-oxoglutarate triggers assembly of active dodecameric Methanosarcina mazei glutamine synthetase

Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 [A] cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on an allosteric binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

microbiology↗

bioRxiv2021

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from {sigma}70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the {Delta}recBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

synthetic biology↗