title: Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates.
abstract: The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Eppinger
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Andreas.Stolz@imb.uni-stuttgart.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Stolz
mesh_terms: D000595:Amino Acid Sequence; D001565:Benzoates / Q000737:chemistry / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006900:Hydroxylation; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D020156:Salicylic Acid / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001565:Benzoates; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; D020156:Salicylic Acid
keywords: gentisate 1,2-dioxygenase; ring-fission dioxygenase; salicylate 1,2-dioxygenase