Abstract
<jats:p> Among the promising microbial platforms to produce value-added products from CO <jats:sub>2</jats:sub> , chemolithoautotrophic bacteria such as <jats:italic toggle="yes">Cupriavidus necator</jats:italic> H16 ( <jats:italic toggle="yes">C. necator</jats:italic> ) stand out due to their ability to fix CO <jats:sub>2</jats:sub> via the Calvin-Benson-Bassham (CBB) cycle. Although <jats:italic toggle="yes">C. necator</jats:italic> has been extensively studied, many enzymes and associated biosynthetic pathways have yet to be identified and fully understood, which is necessary to engineer this microorganism for efficient biotechnological production. One particularly interesting target is the biosynthetic enzymes involved in L-methionine synthesis as this amino acid is currently the only one still produced exclusively from petroleum. In this study, we identified and characterized the enzymatic properties of <jats:italic toggle="yes">Cn</jats:italic> MetXS, a homoserine <jats:italic toggle="yes">O</jats:italic> -succinyltransferase and the key enzyme in the <jats:italic toggle="yes">C. necator</jats:italic> biosynthetic pathway of L-methionine and determined its crystal structure. <jats:italic toggle="yes">Cn</jats:italic> MetXS catalyzes the transsuccinylation of L-homoserine to <jats:italic toggle="yes">O</jats:italic> -succinyl-L-homoserine but exhibits no transacetylase activity. This study verifies <jats:italic toggle="yes">Cn</jats:italic> MetXS as an <jats:italic toggle="yes">O</jats:italic> -succinyltransferase and paves the way for metabolic engineering strategies for the microbial production of L-methionine in <jats:italic toggle="yes">C. necator</jats:italic> . </jats:p>