Characterization and Crystal Structure Elucidation of the l ‐Homoserine O ‐Succinyltransferase MetXS From Cupriavidus necator H16

Among the promising microbial platforms to produce value‐added products from CO 2 , chemolithoautotrophic bacteria, such as Cupriavidus necator H16 ( C. necator ), stand out due to their ability to fix CO 2 via the Calvin–Benson–Bassham (CBB) cycle. Although C. necator has been extensively studied as a biotechnological production host, several of its biosynthetic pathways remain incompletely characterized at the biochemical level. This includes l ‐methionine biosynthesis, for which genome‐based functional annotations of key enzymes have not been validated experimentally. In this study, we identified and characterized Cn MetXS, a homoserine O ‐succinyltransferase involved in l ‐methionine biosynthesis in C. necator . Biochemical characterization demonstrated that Cn MetXS catalyzes the transsuccinylation of l ‐homoserine to O ‐succinyl‐ l ‐homoserine but exhibits no transacetylase activity. Additionally, we determined the apo crystal structure of Cn MetXS at 1.86 Å resolution, revealing a homodimer with a MetX‐G2 active‐site architecture and distinct conformations of the active‐site lid region. Structural analysis and docking suggest that Arg335 stabilizes the succinylated enzyme intermediate and explains the observed succinyl‐CoA specificity. Together, these results establish the function of Cn MetXS and pave the way for metabolic engineering strategies to produce l ‐methionine in C. necator .