title: Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+-dependent oxygenase that cleaves beta-diketones.
abstract: Cupins constitute a large and widespread superfamily of beta-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function. Recombinant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc). Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+ elicited enzymatic catalysis of O2-dependent conversion of various beta-diketone substrates via a nucleophilic mechanism of carbon-carbon bond cleavage. Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 A. Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues. In the 'best-fit' model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 A and 2.08 A, respectively. The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also use a facial triad of histidines for binding of their metal cofactor but promote entirely different substrate transformations.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Leitgeb
authors.forename: Grit D
authors.initials: GD
authors.lastname: Straganz
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D015502:Absorptiometry, Photon; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019117:Burkholderia / Q000201:enzymology*; D002074:Butanones / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D007501:Iron / Q000737:chemistry*; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D002074:Butanones; D007659:Ketones; D007501:Iron; D010105:Oxygenases; C035851:benzoylacetone