title: Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans.
abstract: The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position. Different highly conserved amino acid residues that were supposed to take part in binding and activation of the organic substrates were modified in the SDO by site-specific mutagenesis and the enzyme variants subsequently analyzed for the conversion of salicylate, gentisate and 1-hydroxy-2-naphthoate. The analysis of enzyme variants which carried exchanges in the positions Arg83, Trp104, Gly106, Gln108, Arg127, His162 and Asp174 demonstrated that Arg83 and Arg127 were indispensable for enzymatic activity. In contrast, residual activities were found for variants carrying mutations in the residues Trp104, Gly106, Gln108, His162, and Asp174 and some of these mutants still could oxidize gentisate, but lost the ability to convert salicylate. The results were used to suggest a general reaction mechanism for gentisate-1,2-dioxygenases and to assign to certain amino acid residues in the active site specific functions in the cleavage of (substituted) salicylate(s).
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Eppinger
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze, Sesto Fiorentin, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Ferraroni
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Bürger
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Lenz
authors.initials: L
authors.lastname: Steimer
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Grace
authors.initials: G
authors.lastname: Peng
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze, Sesto Fiorentin, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Briganti
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany. Electronic address: andreas.stolz@imb.uni-stuttgart.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Stolz
mesh_terms: D020561:Alphaproteobacteria / Q000737:chemistry* / Q000201:enzymology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005841:Gentisates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D005841:Gentisates; D009284:Naphthols; D011994:Recombinant Proteins; D012459:Salicylates; D049308:Dioxygenases; C511947:salicylate 1,2-dioxygenase, Pseudaminobacter salicylatoxidans; C026247:gentisate 1,2-dioxygenase; C050175:1-hydroxy-2-naphthoic acid; C010925:2,5-dihydroxybenzoic acid
keywords: Aromatic catabolism; Gentisate dioxygenase; Reaction mechanism; Ring fission