6533b826fe1ef96bd1284cfe

RESEARCH PRODUCT

Characterisation of NO synthases from the alga Klebsormidium nitens

subject

description

Nitric oxide (NO) is an important cellular signaling molecule regulating various physiological processes, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have been measured, although no sequences encoding mammalian NOS have been found in land plants. Interestingly, we identified NOS-like sequences in about twenty algae species. These latter include the filamentous Charophyta green algae Klebsormidium nitens, a biological model to study the early transition step from aquatic algae to land plants. Currently, two NOS were identified in K. nitens genome: i) KnNOS1 which possesses classical mammalian NOS architecture consisting of oxygenase and reductase domains, ii) KnNOS2 displaying a large C-ter extension containing a globin domain. The presence of NOS of great molecular diversity in several algal species while absent from terrestrial plants raises questions about the evolution of this enzyme family in photosynthetic organisms. We initiated the functional characterization of KnNOS by analyzing their primary sequences. From the protein primary sequences, predictive models of the 3D structure of KnNOS were made via AlphaFold and comparison with existing models of mammalian NOS suggested that both KnNOS are functional. Study of the phylogenetic position of KnNOS in comparison to other NOS described in eukaryotes, indicate that both NOS of K. nitens could result from a recent duplication event. The study of their mRNA abundance in response to different abiotic stresses revealed differences suggesting a subfonctionnalisation of these two proteins. In order to study protein partners of KnNOS a pull-down strategy was chosen, requiring the production of recombinant KnNOS1 and KnNOS2. Although the production of KnNOS has not yet been completed, promising results have been obtained in a heterologous yeast system. In parallel, we also built the in silico protein–protein interaction network of human NOS using the BioGRID database and human NOS interaction data. Interestingly, genes encoding orthologs of several of these candidates were found in K. nitens genome. Some of these conserved partners are known to be involved in mammalian NOS regulation and represent interesting candidates for further study. This work has provided new information on the NOS family, notably by characterizing the two NOS isoforms of K. nitens and by highlighting putative differences in their function. Molecular tools developed in this study open the way for a deeper characterization of these proteins and will facilitate future investigations of NO signaling in the green lineage.