Biology subjects

Schuller, J. M.

Publications and source records attributed to Schuller, J. M..

12 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 structure of a 2-MDa chloroplast RNA polymerase reveals unexpected evolutionary complexity

Transcription in chloroplasts depends on the Plastid-Encoded RNA polymerase (PEP), a bacterial-derived enzyme whose catalytic core remains encoded by the highly reduced genome inherited from the cyanobacterial ancestor. In land plants, PEP has roughly doubled in size, expanding into a [~]1 MDa multisubunit machinery through the acquisition of numerous nuclear-encoded subunits. Based on phylogenetic analyses, this added complexity has been widely attributed to the demands of plant terrestrialization. Contrary to this view, we show that in the unicellular green alga Chlamydomonas reinhardtii, PEP assembles into an even larger [~]2 MDa complex containing twelve previously uncharacterized nuclear-encoded subunits (PEPS1-12), representing an RNA polymerase architecture of unprecedented size. A cryo-EM structure at 2.7 [A] resolution reveals that several of these subunits occupy positions analogous to those in land plant PEP, and that metabolic enzyme folds have been repurposed as structural scaffolds stabilizing the highly expanded plastid-encoded core. Despite this, most of the newly identified PEPS subunits lack detectable sequence or structural similarity to their land plant counterparts. These findings demonstrate that PEP complexity is not a hallmark of land plant evolution and may instead reflect, at least in part, the evolutionary entrenchment of additional subunits around an expanded plastid-encoded core. More broadly, they suggest that essential organellar machines can acquire substantial structural complexity that leaves little trace in sequence-based analyses, a pattern consistent with constructive neutral evolution.

molecular biology↗

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↗

bioRxiv2026

Coordination of sequential RNase activities in an ancient molecular machine

The evolutionary mechanisms by which molecular machines incorporate catalytic subunits and coordinate their functions are still poorly understood. Here, we study the molecular evolution of the RNA exosome, an essential RNA-processing machine built from a 9-subunit core (Exo9). Human and yeast Exo9 are catalytically inactive and serve as a recruiting hub for the peripheral RNase Rrp44. The core of modern exosomes descends from an active RNase in Archaea. Using ancestral sequence reconstruction, biochemical and structural characterization, we illuminate how Exo9 evolved from an enzyme to a regulatory hub. The ancestral Exo9 was an active, distributive RNase that already cooperated with Rrp44. Cryo-electron microscopy reveals how RNA-binding modulates the conformation of Exo9, thereby promoting allosteric recruitment of Rrp44. Exo9 begins substrate trimming before the RNA slips past its active site. The RNA can thereby be handed over to Rrp44 to be processed further, which rationalizes the coordination of consecutive RNase activities. We hypothesize that the same allosteric pathway still exists in the human exosome, implying that this mechanism persisted for over a billion years. Our work thus illuminates the evolutionary tinkering that produced a complex molecular machine and a central component of the eukaryotic cell.

evolutionary biology↗

bioRxiv2025

Cryo-EM structure of Chlamydomonas Photosystem I complexedwith the alternative electron donor cytochrome c6

Photosynthetic electron transfer relies on small soluble carriers that shuttle electrons between the cytochrome bf complex and Photosystem I (PSI). While copper-containing plastocyanin (Pc) serves this role in plants, algae and cyanobacteria employ the heme protein cytochrome c (Cyt c) as well. Here we present a cryo-electron microscopy structure of a Cyt c:PSI complex from Chlamydomonas reinhardtii. The Cyt c heme is positioned [~]11 [A] from P700, stabilized by extensive contacts involving the N-terminal domain of PSAF. Importantly, R66 in Cyt c, a key residue in ancestral donors, forms a putative electrostatic contact with PsaB-D623 and participates in a tri-planar {pi}-stacking interaction with nearby aromatic residues. Our findings provide a structural framework for ancestral PSI interactions and illuminate the evolutionary diversification of 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↗

bioRxiv2025

Small ORF16 modulates the Mtr complex activity in Methanosarcina mazei Goe1 depending on the molecular hydrogen availability

Until recently, small open reading frame (sORF)-encoded proteins of fewer than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in conventional detection methodologies. While numerous previously unannotated sORFs have recently been identified in the mesophilic archaeal model system Methanosarcina mazei, the physiological roles of most of their encoded small proteins remain unknown. We report here the functional characterization of sORF16 encoded small protein MtrR (49 amino acids) and show that it localizes oligomerically at the cytoplasmic membrane. There, it interacts with and influences the activity of tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex involved in energy metabolism. In vitro interaction and in vivo copurification assays revealed interactions between MtrR and the Mtr-complex, and microscale thermophoresis showed specific interactions with the MtrA subunit. Mutant strains lacking sORF16 exhibited significantly impaired growth in the presence of molecular hydrogen (H2), irrespective of the carbon source. We posit that by modulating the activity of the Mtr-complex, MtrR enables the archaeon adapt to changing environmental H2 conditions.

microbiology↗

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↗

bioRxiv2023

A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire

Aldehyde oxidoreductases (AOR) are tungsten enzymes catalysing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and utilizes NAD as electron acceptor. Here we reveal that the enzyme forms filaments of repeating AorAB protomers which are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with QM:MM-based modelling for the coordination of the W-co, enables formulation of catalytic mechanism hypothesis that paves the way for further engineering of AOR for applications in synthetic biology and biotechnology.

biochemistry↗

bioRxiv2022

Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex

Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction and some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron transferring proteins EtfA and EtfB. The electron cryo-microscopically (cryo-EM) characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 [A] resolution consists of a mobile EtfAB shuttle located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle contact each other thereby forming the D (dehydrogenase conducting) state. The intermediary Asp37 and Asp139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. A plausible novel B (bifurcation conducting) state with the EtfAB base and shuttle FADs in a productive electron transfer distance was derived by integrating Alphafold2 calculations. Kinetic analysis of enzyme variants shed light on the connection between NAD binding/release and D-to-B state transition. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is based on the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was outlined.

biochemistry↗

bioRxiv2020

How to build a water-splitting machine: structural insights into photosystem II assembly

Biogenesis of photosystem II (PSII), natures water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate with 2.94 [A] resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction. One Sentence HighlightThe high-resolution Cryo-EM structure of the photosystem II assembly intermediate PSII-I reveals how natures water splitting catalyst is assembled, protected and prepared for photoactivation by help of the three assembly factors Psb27, Psb28 and Psb34.

plant biology↗

bioRxiv2020

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the rings lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

plant biology↗