Structural and biochemical characterisation of N2-assimilation system and its regulation in thermophilic methanogenic archaea
|Authors:||Maslac, Nevena||Supervisor:||Wagner, Tristan||1. Expert:||Wagner, Tristan||Experts:||Milton, Ross D.||Abstract:||
Nitrogen gas can only be biologically reduced to ammonia by diazotrophic microorganisms. Among these microbes, methanogens and anaerobic methanotrophic archaea represent the sole known archaeal diazotrophs, and they have been found to actively participate in nitrogen fixation within anaerobic environments such as marine and marsh sediments. The nitrogenase enzyme, catalysing the reduction, also serves as a model for developing effective and sustainable catalysts for industrial ammonia production in the Haber-Bosch process. However, numerous questions regarding the molecular mechanisms underlying this catalysis remain unanswered, and the structure of archaeal nitrogenases, proposed to be more ancient than bacterial, remains elusive.
In Chapter 2, we investigated the adaptation and cellular response of the thermophile Methanothermococcus thermolithotrophicus to N2-fixation using differential transcriptomics. Our findings reveal that M. thermolithotrophicus would employ multiple pathways for nitrogen acquisition and a targeted response for energy saving through the downregulation of genes involved in transcription and translation. Chapter 3 details the structures of nitrogenase reductase from three marine Methanococcales. Notably, the structures obtained from M. thermolithotrophicus and Methanocaldococcus infernus represent the first natively purified and solved nitrogenase reductases from methanogens. In Chapter 4, we present the first high-resolution structure of archaeal nitrogenase from M. infernus in both resting and mixed turnover states, supporting the existence of a common reduction mechanism shared by all nitrogenases. Additionally, we unveiled the molecular basis of the posttranslational regulation of archaeal N2-fixation by characterising an inhibitory closed circular complex mediated by PII family proteins.
To address two long-standing questions related to molybdenum nitrogenase, Chapters 5 and 6 combine structural biology with the robust genetic system of Azotobacter vinelandii. By creating nitrogenase variants, we clarified the functional and structural role of an additional metal binding site at a dimeric protein interface and demonstrated negative cooperativity in the Mo-dependent nitrogenase.
In summary, this thesis delves into characterisation of diazotrophic microbes on multiple molecular levels, with an emphasis on nitrogen assimilation systems in (hyper)thermophilic methanogens. While shedding light on the intricate mechanisms underlying these processes, it offers valuable insights into nitrogen fixation in these ancient organisms.
|Keywords:||nitrogenase; nitrogen fixation; methanogens; hyperthermophiles; X-ray crystallography||Issue Date:||10-Aug-2023||Type:||Dissertation||DOI:||10.26092/elib/2553||URN:||urn:nbn:de:gbv:46-elib72431||Research data link:||http://www.ncbi.nlm.nih.gov/bioproject/PRJEB53446
|Institution:||Universität Bremen||Faculty:||Fachbereich 02: Biologie/Chemie (FB 02)|
|Appears in Collections:||Dissertationen|
checked on Dec 10, 2023
checked on Dec 10, 2023
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