Biology subjects

Rebelein, J. G.

Publications and source records attributed to Rebelein, J. G..

6 recordsLinked to original sources

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

Promiscuous NAD-dependent dehydrogenases enable efficient bacterial growth on the PET monomer ethylene glycol

Ethylene glycol is widely used as antifreeze agent and monomer of the ubiquitous plastic PET (polyethylene terephthalate). Its global production amounts to more than 50 million tons per year, and it constitutes an environmental pollutant of increasing concern. Although it is generally accepted that bacteria oxidize ethylene glycol to use it as growth substrate, the enzymes involved in this process are not well understood. Here we show that the soil bacterium Paracoccus denitrificans is able to assimilate ethylene glycol efficiently via NAD-dependent alcohol and aldehyde dehydrogenases. Using comparative proteome analysis, we identify a previously unknown gene cluster that is strongly expressed in the presence of ethylene glycol. We report the kinetic parameters and cryo-EM structures of EtgB and EtgA, the key enzymes encoded by this etg gene cluster. These novel biocatalysts pave the way for more efficient biotechnological conversion of ethylene glycol. We furthermore show that the transcriptional activator EtgR controls expression of the etg gene cluster. Directed evolution of P. denitrificans on ethylene glycol results in faster growing strains, which is enabled by increased activities of EtgB and EtgA. Bioinformatic analysis reveals that the etg gene cluster and variants thereof are widely distributed among Proteobacteria, suggesting a previously underappreciated role of NAD-dependent dehydrogenases in microbial ethylene glycol assimilation.

microbiology↗

bioRxiv2023

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and, hence, are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterised the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterised by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of {Delta}fdxC and {Delta}fdxN strains, in conjunction with differing plasmid complementation behaviours of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation. ImportanceNitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. Production of ammonia by diazotrophic organisms, harbouring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, though we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterised the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centres. Additionally, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviours.

biochemistry↗

bioRxiv2023

CO2 Reduction by the Iron Nitrogenase Competes with N2 Fixation Under Physiological Conditions

Nitrogenases are the only known enzymes that reduce molecular nitrogen (N2) to ammonia. Recent findings have demonstrated that nitrogenases also reduce the greenhouse gas carbon dioxide (CO2), suggesting CO2 to be a competitor of N2. Intriguingly, nitrogenase isoforms (i.e., molybdenum (Mo), vanadium and iron (Fe) nitrogenase) differ significantly in their ability to reduce CO2, but the mechanisms underlying these differences remain elusive. Here, we study the competing reduction of CO2 and N2 by the two nitrogenases of Rhodobacter capsulatus, the Mo and Fe nitrogenase. Analyzing their full CO2 reduction product spectrum in vitro, we find the Fe nitrogenase almost three-fold more efficient in CO2 reduction than the Mo isoform. Furthermore, the in vitro competition experiments reveal the Fe nitrogenase to be profoundly less selective for the reduction of N2 than the Mo nitrogenase. We observe the same effects in vivo, where adding CO2 drastically increases the doubling times of diazotrophically grown R. capsulatus strains that rely on the Fe nitrogenase. The Fe nitrogenase-dependent R. capsulatus strains reduce CO2 to methane under physiological conditions, highlighting the potential of the Fe nitrogenase for the biotechnological conversion of CO2 into value-added compounds. Furthermore, both products are secreted into the surrounding, potentially influencing the composition of microbial communities in Mo-deficient environments.

biochemistry↗

bioRxiv2023

Structural Insights into the Iron Nitrogenase Complex

Nitrogenases are best known for catalysing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the nitrogenase family the iron nitrogenase is the isozyme with the highest wildtype activity for the reduction of CO2, but the molecular architecture facilitating these activities remained unknown. Here, we report a 2.35-[A] cryogenic electron microscopy structure of the Fe nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate]-cluster in the active site. The enzyme complex suggests that the AnfG-subunit is involved in cluster stabilisation, substrate channelling and confers specificity between nitrogenase reductase and catalytic components. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intra-subunit communication and thus the nitrogenase mechanism.

biochemistry↗

bioRxiv2023

Methane formation driven by light and heat prior to the origin of life

Methane is a potent greenhouse gas, which likely enabled the evolution of life by keeping the early Earth warm. Here, we demonstrate new routes towards abiotic methane formation under early-earth conditions from methylated sulfur and nitrogen compounds with prebiotic origin. These compounds are demethylated in Fenton reactions governed by ferrous iron and reactive oxygen species, produced by light and heat in aqueous environments. The reactions generate methyl radicals and ultimately release methane and ethane. Organic iron chelators enhance reaction rates and recycle ferric to ferrous complexes via ligand-to-metal charge transfer, establishing a light-driven iron redox cycle. This abiotic reaction facilitates methane and ethane formation across Earths humid realm, thereby shaping the chemical evolution of the atmosphere prior to the origin of life and beyond. One-Sentence SummaryUnder suboxic and anoxic conditions, iron and reactive oxygen species drive the global formation of methane in aqueous environments.

evolutionary biology↗