Biochemical characterization and mutational analysis of the mononuclear non-heme Fe2+ site in Dke1, a Cupin-type dioxygenase from Acinetobacter johnsonii

Stefan Leitgeb , Grit D Straganz , Bernd Nidetzky (1)

1 TU Graz - Graz University of Technology [Graz]

β-Diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1 protomer adopts a single-domain β-barrel fold characteristic of the Cupin superfamily of proteins and features a mononuclear non-heme Fe2+ centre where a triad of histidines, His-62, His-64, and His-104, coordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-heme Fe2+, we replaced each histidine individually by Glu and Asn and compared binding of Fe2+ and four non-native, catalytically inactive metals to purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+, Zn2+) experiments revealed the presence of two broadly specific metal binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 μM (site I) and ≈ 3 x 102 μM (site II). Each mutation except the substitution of His-104 by Asn disrupted binding of Fe2+, but not that of the other divalent metal ions, at site I while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring substitutions by Glu were completely inactive and not functionally complemented by external Fe2+. The Fe2+ catalytic centre activity (kcat) of mutants with Asn substitution of His-62 and His-104 was decreased 140- and 220-fold, respectively, as compared to the kcat value of 8.5 s-1 for the wild-type enzyme in the reaction with 2,4-pentanedione. The His-64→Asn mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore, coordination of Fe2+ by Dke1 requires an uncharged metallocentre, and 3 His ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.

Mots clés: Life Sciences

Stefan Leitgeb, Grit D Straganz, Bernd Nidetzky. Biochemical characterization and mutational analysis of the mononuclear non-heme Fe2+ site in Dke1, a Cupin-type dioxygenase from Acinetobacter johnsonii. Biochemical Journal, 2009, 418 (2), pp.403-411. ⟨10.1042/BJ20081161⟩. ⟨hal-00479042⟩