In silico structural and functional characterisation of two hypothetical proteins from the plant‑associated bacterium Kosakonia cowanii F52.

Kosakonia cowanii promotes plant growth under salt stress, but many of its hypothetical proteins (HPs) remain uncharacterised. We screened 218 HPs from K. cowanii F52 using sequential filters for physicochemical stability, subcellular localisation, and conserved domains. Four candidates were modelled with AlphaFold2 and validated by stereochemical and global geometry scores. Two HPs, MHQ1860809.1 and MHQ1862734.1, were selected based on combined global and per-residue model-quality metrics for further functional annotation via structural similarity searches, Gene Ontology (GO), and KEGG pathway analysis. Molecular docking of NAD⁺ was subsequently performed on MHQ1860809.1 to evaluate cofactor binding. MHQ1860809.1 is predicted to adopt a canonical Rossmann fold with a conserved NAD(P)-binding pocket; docking was consistent with NAD⁺ binding (Vina score -7.8 kcal/mol). GO predicted oxidoreductase activity (GO:0016646) and proline metabolic process (GO:0006560). MHQ1862734.1 is predicted to fold into an ARM-repeat superhelix, lacks any ligand-binding cavity, and is structurally similar to importin-β and AP-2 adaptor subunits. However, no EC numbers or KEGG orthologies could be assigned. MHQ1860809.1 is therefore structurally consistent with a putative NAD(P)-dependent oxidoreductase potentially involved in proline biosynthesis, whereas MHQ1862734.1 adopts an ARM-repeat fold of unknown function. These findings generate specific, testable hypotheses and provide a foundation for future experimental work on the molecular basis of K. cowanii plant growth-promoting activity.