Biology subjects

Lemaire, O. N.

Publications and source records attributed to Lemaire, O. N..

4 recordsLinked to original sources

bioRxiv2024

Snapshots of acetyl-CoA synthesis, the final step of CO2 fixation in the Wood-Ljungdahl pathway

In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process ending with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO2 fixation in anaerobic organisms. One-Sentence Summary: Structural description of key states of CO2 fixation by the carbon monoxide dehydrogenase/acetyl-CoA synthase complex.

biophysics↗

bioRxiv2024

Carbon monoxide-driven bioethanol production operates via a tungsten-dependent catalyst

Microbial alcohol production from waste gasses is a game changer for sustainable carbon cycling and remediation. While the biotechnological process employing Clostridium autoethanogenum to transform syngas (H2/CO2/CO) is blooming, the reactions involved in ethanol biosynthesis remain to be demonstrated. Here, we experimentally showed that ethanol production initiates via a tungsten-dependent aldehyde:ferredoxin oxidoreductase (AFOR), which reduces acetate to acetaldehyde. Such an unfavourable reaction has often been considered unsuitable for a biological process. To answer this riddle, we demonstrated that the thermodynamic pull of CO-oxidation and ethanol synthesis is crucial for triggering acetate reduction. The experimental setup performed with native CO-dehydrogenase and AFOR highlighted the key role of ferredoxin in stimulating the activity of both metalloenzymes and electron shuttling. The crystal structure of holo AFOR refined to 1.59-[A] resolution, together with its biochemical characterisation, provides new insights into the reaction mechanism and the specificities of this enzyme fundamental to sustainable biofuel production.

biochemistry↗

bioRxiv2024

F420 reduction as a cellular driver for anaerobic ethanotrophy

The anaerobic ethane oxidation performed by seafloor archaea and sulfate-reducing partner bacteria involves largely uncharted biochemistry. This study deciphers the molecular basis of the CO2-generating steps by characterizing the native archaeal enzymes isolated from a thermophilic enrichment culture. While other microorganisms couple these steps to ferredoxin reduction, we found that the CO-dehydrogenase and the formylmethanofuran-dehydrogenase are bound to an F420-reductase module. The crystal structures of these multi-metalloenzyme complexes revealed a [4Fe-4S]-cluster networks electronic bridges coupling C1-oxidation to F420-reduction. Accordingly, both systems exhibit robust F420-reductase activities, which are not detected in methanogenic or methanotrophic relative organisms. We speculate that the whole catabolism of these archaea is reoriented towards F420-reduction, which facilitates the electron transfer to the sulfate-reducing partner, therefore representing the driving force of ethanotrophy.

biochemistry↗

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↗