Biology subjects

Müller, M.-C.

Publications and source records attributed to Müller, M.-C..

3 recordsLinked to original sources

bioRxiv2025

Molecular mechanism of nitrogenase sequestration by a PII-protein couple

The microbial process of N2-fixation is crucial for the planetary nitrogen cycle and biosphere, but requires substantial cellular energy resources. Here, we solved how energy-limited anaerobes regulate on-demand N2-fixation by sequestering their nitrogenase through the PII-nitrogen regulatory proteins NifI1 and NifI2. The nitrogenase was directly isolated from a methanogenic archaeon, with NifI proteins tightly bound. The crystal structure of the inhibited form, refined to 2.3-[A] resolution, reveals a supercomplex in which three NifI1,2 units made of a NifI1,2 heterohexamer captured three nitrogenases via tentacular T-loops. Additional structural information confirmed that NifI1,2 sits at the nitrogenase reductase-binding site, preventing N2-reduction. The presence of MgATP and 2-oxoglutarate releases NifI1,2 from the nitrogenase core via a conformational switch of the T-loops, provoking a steric repulsion and loss of contacts. The overall molecular depiction corroborates previous genetic, biochemical, and biophysical experiments, proposing that NifI1,2 disrupts the dynamic nitrogenase-reductase association, thereby interfering with electron delivery for N2-fixation and preventing ATP consumption. While ligand-binding mode and the T-loop conformational switch are expected to be conserved among NifI1,2-utilisers, the association mode with the nitrogenase comes in different flavours as a few substitutions in NifI2 break NifI1,2 intramolecular dimerisation in Methanosarcinales species, readjusting the supercomplex without altering the inhibition mechanism. With the NifI1,2 allosteric control dependent on the alarmone 2-oxoglutarate, anaerobes can effectively balance nitrogen-acquisition versus energy-expenditure, a regulatory switch that might be primitive and has been progressively lost in non-energy-limited aerobes, but could inspire biotechnological engineering to optimise ammonia bioproduction.

biochemistry↗

bioRxiv2025

Atomic resolution structures of key enzyme MCR in anaerobic methanotrophy reveal novel and extensive post-translational modifications.

Anaerobic methanotrophic archaea (ANME) are crucial to planetary carbon cycling. They oxidise methane in anoxic niches by transferring electrons directly to nitrate or metal oxides and alternatively to sulfate-reducing bacteria. Due to their physiological complexity, no ANME species have been isolated, hampering the biochemical investigation of the enzymatic processes involved in anaerobic methane oxidation. To study the methane-capturing enzyme of these microorganisms, we circumvented the isolation barrier by exploiting microbial enrichments of freshwater nitrate-reducing ANME-2d grown in bioreactors, and marine ANME-2c in syntrophy with bacterial partners. The crystal structures of their Methyl-Coenzyme M Reductases (MCRs), refined to true atomic resolution, provided the most precise image of the enzyme to date. Despite their physiological differences, these ANMEs have extremely conserved MCR structures, similar to homologs from methanogenic Methanosarcinales, rather than the phylogenetically distant MCR of ANME-1 isolated from Black Sea mats. The three studied MCRs are highly modified, with seven post-translational modifications. Among them was a novel 3(S)-methylhistidine on the {gamma}-chain of both ANME-2d MCRs. Labelling with gaseous krypton did not reveal any internal channels that would facilitate alkane diffusion to the active site as observed in the ethane-specialized enzyme. Based on our data, the methanotrophic MCRs should follow the same radical reaction mechanism proposed for the methane-generating homologues. The described pattern of post-translational modifications underscores the importance of native purification as a powerful approach to discovering intrinsic enzymatic features in uncultivated microorganisms existing in nature.

microbiology↗

bioRxiv2023

Differences in the regulation mechanisms of the glutamine synthetase from methanogenic archaea unveiled by structural investigations

Glutamine synthetases catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be tightly regulated to fit cellular needs. While their catalytic mechanisms and regulation are well-characterized in bacteria and eukaryotes, only limited knowledge exists about the archaeal representatives. Here, we natively purified the glutamine synthetases type I- from Methanothermococcus thermolithotrophicus and Methermicoccus shengliensis, two thermophilic methanogens belonging to different orders. Biochemical investigations combined with X-ray crystallography unveiled the first structures of archaeal glutamine synthetases and highlighted differences in their regulation. The enzyme from M. thermolithotrophicus is inactive in its resting state and employs 2-oxoglutarate as an on-switch. The 2-oxoglutarate acts as a sensor of cellular nitrogen deficiency, and its reported cellular concentration remarkably overlays with that required for the enzyme activation. Its binding to an allosteric pocket leads to the reconfiguration of the active site and promotes a catalytically competent state. The homolog from M. shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited by glutamine, as shown for bacterial homologs. The glutamine inhibition depends on a key arginine residue from the Asp50-loop. The arginine is substituted by a glycine in M. thermolithotrophicus, abolishing the inhibitory effect. While the effectors are surprisingly different, the molecular switch controlling the glutamine synthetase activity is fundamentally the same and depends on the correct positioning of the Asp50-loop and a catalytic arginine. Residue conservation suggests that both regulation mechanisms are widespread and not mutually exclusive across archaea.

biochemistry↗