Structural Superimposition

Abstact

Sulfoquinovose dioxygenase (SqoD) enables bacterial carbon assimilation from the abundant sulfosugar sulfoquinovose (SQ) by Fe(ii)/alpha-ketoglutarate (alphaKG)-dependent C-S bond cleavage. Here we report crystal structures of the Marinobacterium aestuarii enzyme (MaSqoD) in multiple states with inert Mn(2+) in place of Fe(2+) (SQ-bound; Mn(2+).alphaKG; Mn(2+).alphaKG.SQ; Mn(2+).succinate), together with steady-state and pre-steady-state kinetics that link the structures with kinetically-inferred intermediates. The X-ray crystal structures show a canonical 2-His-1-carboxylate core metal center with SQ recognition via a mainly neutral network (Gln120, Trp253, backbone carbonyl of Ala185, and backbone amides of Ala89/Met118). Substrate binding triggers a hexacoordinate octahedral-to-pentacoordinate change at the metal center, unveiling a vacant site for O(2) in the fully assembled Mn(2+).alphaKG.SQ complex and thereby curbing uncoupled reactions. Pre-steady-state stopped-flow data support the canonical Fe(iv)[double bond, length as m-dash]O chemistry of the dioxygenase and reveal an additional intermediate consistent with an enzyme-bound alpha-hydroxysulfonate or 6-dehydroglucose species. Together, these findings define the structural and mechanistic basis of sulfolytic SQ catabolism and illuminate the functional repertoire of Fe(ii)/alphaKG-dependent dioxygenases in organosulfur carbon assimilation.