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The salicylate 1,2‐dioxygenase as a model for a conventional gentisate 1,2‐dioxygenase: crystal structures of the G106A mutant and its adducts with gentisate and salicylate
Author(s) -
Ferraroni Marta,
Matera Irene,
Bürger Sibylle,
Reichert Sabrina,
Steimer Lenz,
Scozzafava Andrea,
Stolz Andreas,
Briganti Fabrizio
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12173
Subject(s) - chemistry , dioxygenase , corynebacterium glutamicum , stereochemistry , oxidoreductase , enzyme , mutant , biochemistry , gene
The salicylate 1,2‐dioxygenase ( SDO ) from the bacterium P seudaminobacter salicylatoxidans BN 12 is a versatile gentisate 1,2‐dioxygenase ( GDO ) that converts both gentisate (2,5‐dihydroxybenzoate) and various monohydroxylated substrates. Several variants of this enzyme were rationally designed based on the previously determined enzyme structure and sequence differences between the SDO and the ‘conventional’ GDO from Corynebacterium glutamicum . This was undertaken in order to define the structural elements that give the SDO its unique ability to dioxygenolytically cleave (substituted) salicylates. SDO variants M 103 L , G 106 A , G 111 A , R 113 G , S 147 R and F 159 Y were constructed and it was found that G 106 A oxidized only gentisate; 1‐hydroxy‐2‐naphthoate and salicylate were not converted. This indicated that this enzyme variant behaves like previously known ‘conventional’ GDO s. Crystals of the G 106 A SDO variant and its complexes with salicylate and gentisate were obtained under anaerobic conditions, and the structures were solved and analyzed. The amino acid residue Gly106 is located inside the SDO active site cavity but does not directly interact with the substrates. Crystal structures of G 106 A SDO complexes with gentisate and salicylate showed a different binding mode for salicylate when compared with the wild‐type enzyme. Thus, salicylate coordinated in the G 106 A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that was formed between T rp104 and A sp174 in the G 106 A variant. It is proposed that this type of unproductive substrate binding might generally limit the substrate spectrum of ‘conventional’ GDO s. Database Structural data are available in the Protein Data Bank databases under the accession numbers 3NST , 3NWA , 3NVC