Biology subjects

Bolte, P.

Publications and source records attributed to Bolte, P..

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

Molecular basis of N2 fixation in a hyperthermophilic archaeon

Exploring the natural diversity of phylogenetically distant nitrogenases is crucial for gaining new insights into the mechanism of atmospheric N2 fixation and unlocking biotechnological developments in sustainable ammonia production. Here, we investigated the N2-fixing system of Methanocaldococcus infernus, a deep-sea hyperthermophilic archaeon growing diazotrophically at 92 {degrees}C. This natively isolated nitrogenase has a melting temperature close to the water boiling point, with an extrapolated specific activity superior to mesophilic homologues. The crystal structures obtained at near-atomic resolution present the most simplified known nitrogenase, harbouring strategic hot spots for thermostability. It combines the structural traits of all three known nitrogenase isoforms, reinforcing the postulate that the archaeal enzyme predates modern versions. In contrast to structural homologues, the electron-transferring metallocofactor "P-cluster" is trapped in a rare state awaiting electron delivery, providing a detailed picture of the physiological state. The active site, harbouring the FeMo-cofactor catalyst, exhibits a mixture of the resting and "turnover" states, previously described solely in the bacterial vanadium and iron-only nitrogenases. Therefore, these results unify a mechanistic principle of all nitrogenases and highlight the advantages of the hyperthermostable nature of the archaeal enzyme, opening new avenues for further understanding of how nature splits the N2 triple bond.

biochemistry↗

bioRxiv2024

Full-length Cryptochrome 1 in the outer segments of the retinal blue cone photoreceptors in humans and great apes suggests a role beyond transcriptional repression

Mammalian cryptochrome 1 (CRY1) is a central player in the circadian transcription-translation feedback loop, crucial for maintaining a roughly 24-hour rhythm. CRY1 was suggested to also function as blue-light photoreceptor in humans and has been found to be expressed at the mRNA level in various cell types of the inner retina. However, attempts to detect CRY1 at the protein level in the human retina have remained unsuccessful so far. Using various C-terminal specific antibodies recognizing full-length CRY1 protein, we consistently detected selective labelling in the outer segments of short wavelength-sensitive (SWS1, "blue") cone photoreceptor cells across human, bonobo, and gorilla retinae. No other retinal cell types were stained, which is in contrast to what would be expected of a ubiquitous clock protein. Subcellular fractionation experiments in transfected HEK cells using a C-terminal specific antibody located full-length CRY1 in the cytosol and membrane fractions. Our findings indicate that human CRY1 has several different functions including at least one non-clock function. Our results also raise the likely possibility that several different versions of CRY1 exists in humans. We suggest that truncation of the C-terminal tail, maybe to different degrees, may affect the localization and function of human CRY1.

cell biology↗