Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans.
The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.
Hintner
Jan-Peter
JP
Institute for Microbiology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Reemtsma
Thorsten
T
Stolz
Andreas
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D020560:Proteobacteria / Q000378:metabolism*; D020156:Salicylic Acid / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D004269:DNA, Bacterial; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; D020156:Salicylic Acid