Biology subjects

Hofmann, E.

Publications and source records attributed to Hofmann, E..

11 recordsLinked to original sources

bioRxiv2026

The -hydrogenase SoHydAB of Shewanella oneidensis - An Atypical M2(D)-type Enzyme that Drives Formate Oxidation

The gamma-proteobacterium Shewanella oneidensis harbours one [NiFe]- and one [FeFe]-hydrogenase. This paper presents the first biochemical characterisation of SoHydAB, a M2(D)-type [FeFe]-hydrogenase consisting of a small subunit featuring a twin-arginine-targeting motif (TAT) and a large subunit harbouring the active site cofactor and two accessory iron-sulfur clusters. There are three main reasons to investigate this enzyme. Firstly, S. oneidensis is a highly studied, metabolically complex, and widely utilised organism, especially in the context of wastewater treatment, CO2 conversion, and microbial fuel cells, including H2-producing systems. Advanced understanding of its hydrogen metabolism can improve biotechnological applications. Secondly, S. oneidensis has been a mainstay of hydrogenase research for decades as its enzymes HydE, F and G are the most widely studied and utilised maturases - enzymes assembling the active site cofactor of [FeFe]-hydrogenases. Lastly, while enzymes like NdHydAB from Nitratidesulfovibrio vulgaris (formerly DdHydAB, Desulfovibrio vulgaris) with high similarity to SoHydAB have been characterised extensively in the past, the metabolic context of the enzyme in Shewanella is different from Nitratidesulfovibrio. While NdHydAB receives electrons from ferredoxin, SoHydAB accepts electrons from the menaquinone pool of the outer membrane while being a soluble enzyme in the periplasm. This is facilitated by interaction with a membrane-integrated cytochrome b transcribed from the same operon. Oxidation of the menaquinone pool drives oxidation of formate toward CO2 via formate dehydrogenase. This study therefore lays the groundwork for understanding H2-dependent CO2 turnover, a new type of metabolic niche for [FeFe]-hydrogenases, while simultaneously improving our understanding of a widely used platform organism.

biochemistry↗

bioRxiv2026

A histidine-mediated, pendulum-like proton transport mechanism is required for the high catalytic activity of -hydrogenases

[FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.

biochemistry↗

bioRxiv2026

Direct Binding of Cysteine-367 Thiolate to the Active Site of the -Hydrogenase from Clostridium beijerinckii in the O2-stable State

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

biochemistry↗

bioRxiv2026

Prophylactic and therapeutic antiviral effects of the influenza A defective interfering particle OP7 in human lung epithelial cells in vitro

Defective interfering particles (DIPs) derived from the influenza A virus (IAV) are a promising antiviral agent due to their strong antiviral efficacy demonstrated in various animal models. OP7 is an unconventional IAV DIP with multiple point mutations in the viral RNA (vRNA) of genome segment 7, as opposed to the large internal genomic deletions typically found in conventional IAV DIPs. Further, OP7 showed an even higher interfering efficacy than conventional DIPs. However, the inhibitory effect of OP7 on standard virus (STV) replication has primarily been investigated in Madin-Darby Canine Kidney (MDCK) cells, which lack a functional myxovirus resistance (Mx)-mediated antiviral activity against IAV. In this study, we examined the antiviral activity and mechanism of antiviral action of OP7 in an interferon (IFN)-competent human lung carcinoma cell line (Calu-3) in vitro. We performed STV and OP7 co-infection experiments using a variety of infection conditions and measured the time-resolved dynamics in viral titer, vRNA, protein level, and host cell gene expression. We observed that OP7 co-infection results in enhanced type I IFN responses and markedly reduced infectious virus release, even at low doses. Additionally, we found that at a high STV multiplicity of infection (MOI), the replication interference of OP7, suppressing the replication of STV vRNA, appears to be the dominant mechanism of its antiviral action. At a low MOI, however, IFN induction seems to be more important. Furthermore, we examined the efficacious co-infection time window for potential prophylactic and therapeutic antiviral treatment. We observed an antiviral effect exerted by OP7 infection for up to seven days before STV infection and up to 24 hours after STV infection. Together, these findings demonstrate that OP7 is a potent antiviral DIP. Therefore, this work supports the further development of OP7 as a therapeutic and prophylactic antiviral agent.

molecular biology↗

bioRxiv2026

Loss of cardiomyocyte eukaryotic elongation factor 1 A2 in adult mice triggers cardiomyopathy due to defective proteostasis

Eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to ribosomes but also has additional, non-canonical functions. Mammals express two paralogs: eEF1A1 is ubiquitous, whereas eEF1A2 is confined to adult cardiomyocytes, skeletal myocytes, and neurons. Mutations in EEF1A2 cause cardiomyopathy, but underlying mechanisms remain unclear. Using adult, cardiomyocyte-specific Eef1a2 knock-out (Eef1a2-cKO) and Eef1a1/Eef1a2 double knock-out mice, we show that Eef1a2-cKO animals develop cardiomyopathy with increased mortality, systolic dysfunction, and fibrosis, despite unchanged global protein synthesis, while double knock-out mice die early in a sudden manner. Multi-omics analyses reveal post-transcriptional upregulation of ribosomal proteins and translational regulators in both models. Eef1a2-cKO hearts accumulate autophagosomes and protein aggregates, indicating defective autophagy. Mechanistically, we found that eEF1A2 functions as a chaperone supporting protein folding and proteostasis in cardiomyocytes. Early Rapamycin treatment (mTORC1 inhibition) normalizes systolic heart function and survival in Eef1a2-cKO mice and clears autophagosomes and protein aggregates. Thus, eEF1A2 maintains cardiac proteostasis, and mTORC1 inhibition may represent a therapeutic strategy for patients with EEF1A2 mutations.

physiology↗

bioRxiv2025

Understanding PebS-Ferredoxin Recognition: A Structural Perspective on Viral and Host Redox Partners

Cyanophages play a critical role in shaping host metabolism during infection by encoding auxiliary metabolic genes (AMGs), including enzymes involved in phycobilin biosynthesis. PebS (phycoerythrobilin synthase) is a phage-encoded ferredoxin-dependent biliverdin reductase (FDBR) that catalyses the two-step reduction of biliverdin IX (BV) to phycoerythrobilin (PEB). Although high-resolution structures are available for both PebS and its electron donor ferredoxin (Fd), the structural details governing their interaction remain unclear. This study leverages the well-characterised structural models of PebS and a host-like ferredoxin from Thermosynechococcus elongatus (Te-Fd), whose NMR structure provides a reliable basis for probing protein- protein interactions. Using [{superscript 1}N]-labelled, gallium-substituted Te-Fd, we employed NMR spectroscopy to monitor chemical shift perturbations upon binding, enabling us to probe the interaction interface in solution. Based on these data, we conducted protein-protein docking with HADDOCK to predict the interaction interface between Te-Fd and PebS. PebS variants designed to disrupt this interface did indeed show corresponding alterations in enzymatic efficiency and product formation as determined by time resolved UV/Vis spectroscopy and HPLC analyses. Utilizing the Fd encoded in the cyanophage PSSM2-Fd in our assays, we could observe a significantly improved catalytic activity, suggesting an coevolution of phage enzyme and electron donor. A comparison of the two available X-ray structures of Te-Fd and PSSM2-Fd with an alphafold-model of the Fd of the natural host Prochlorococcus NATL1A (NATL1A-Fd) also supports this evolutionary adaptation and the role of both PSSM2-Fd and PebS as AMGs involved in viral infection by PSSM2.

molecular biology↗

bioRxiv2025

Structural insights into late-stage photosystem II assembly by Psb32

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In particular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the Mn4O5Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates from Thermosynechococcus vestitus BP-1. In contrast to previous studies, the resulting monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn4O5Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precedes the incorporation and photoactivation of the Mn4O5Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing.

plant biology↗

bioRxiv2024

The activation of Chlamydomonas reinhardtii alpha amylase 2 by glutamine requires its N-terminal ACT domain

The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of Chlamydomonas reinhardtii that we captured by a cyclic guanosine monophosphate-(cGMP-) affinity chromatography approach, namely soluble starch synthase STA3, starch branching enzyme SBE1 and -amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzymes N-terminal aspartate kinase-chorismate mutase- tyrA (ACT) domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules, but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N- and C metabolism at the enzymatic level, increasing the liberation of C-skeletons from starch when high Gln levels signal an abundance of assimilated N.

plant biology↗

bioRxiv2022

On the evolution of the plant phytochrome chromophore

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria and fungi. Land plant phytochromes use phytochromobilin (P{Phi}B) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IX (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to P[FE]B by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only P[FE]B biosynthesis related genes (HY2). The HY2 of the early diverging streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) was already indirectly indicated to be involved in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in E. coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we were able to confirm the product and to identify intermediates of the reaction. Site-directed mutagenesis revealed two aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a P{Phi}B-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of two additional members of the HY2 lineage enabled us to define two distinct clades, the PCB-HY2 and the P{Phi}B-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

plant biology↗

bioRxiv2021

The mating type transcription factor MAT1-1-1 from the fungal human pathogen Aspergillus fumigatus: synthesis, purification, and crystallization of the DNA binding domain.

Mating type (MAT) loci are the most important and significant regulators of sexual reproduction and development in ascomycetous fungi. Usually, they encode two transcription factors (TFs), named MAT1-1-1 or MAT1-2-1. Mating-type strains carry only one of the two TF genes, which control expression of pheromone and pheromone receptor genes, involved in the cell-cell recognition process. The present work presents the crystallization for the alpha1 (1) domain of MAT1-1-1 from the human pathogenic fungus Aspergillus fumigatus (AfMAT1-1-1). Crystals were obtained for the complex between a polypeptide containing the 1 domain and DNA carrying the AfMAT1-1-1 recognition sequence. A streak seeding technique was applied to improve native crystal quality, resulting in diffraction data to 3.2 [A] resolution. Further, highly redundant data sets were collected from the crystals of selenomethionine-substituted AfMAT1-1-1 with a maximum resolution of 3.2 [A]. This is the first report of structural studies on the 1 domain MAT regulator involved in the mating of ascomycetes. SynopsisAn optimized purification and crystallization protocol together with initial X-ray datasets are described for this mating type transcription factor from human pathogenic fungus Aspergillus fumigatus.

microbiology↗

bioRxiv2020

Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity

Cryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta. We show GtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP {beta}-subunit (PmCpeB) of Prochlorococcus marinus MED4. Based on the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colorful complex in vitro and in vivo produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.

biochemistry↗