Spontaneous release of fluoride during the dioxygenolytic cleavage of 5-fluorosalicylate by the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans BN12

• Published in: FEMS microbiology letters Vol. 363; no. 1; p. fnv211
• Main Authors: Eppinger, Erik, Bürger, Sibylle, Stolz, Andreas
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2016
• Subjects: Absorbance; Chromatography; Chromatography, High Pressure Liquid; Cleavage; Conversion; Dioxygenases - genetics; Dioxygenases - metabolism; E coli; Enzymes; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorides; Fluorides - metabolism; Gene Expression; Gentisate 1,2-dioxygenase; High performance liquid chromatography; Liquid chromatography; Magnetic Resonance Spectroscopy; Mass spectrometry; Mass spectroscopy; Microbiology; NMR; Nuclear magnetic resonance; Phyllobacteriaceae - enzymology; Phyllobacteriaceae - genetics; Phyllobacteriaceae - metabolism; Propionic acid; Reaction products; Salicylates - metabolism; Salicylic acid; Spectrophotometry; Stereoisomerism; Stereoisomers; fluorinated aromatics; gentisate 1,2-dioxygenase; salicylate 1,2-dioxygenase; biodegradation; ring-fission dioxygenases; Pseudaminobacter salicylatoxidans
• ISSN: 1574-6968; 0378-1097; 1574-6968
• DOI: 10.1093/femsle/fnv211

The alpha-Proteobacterium Pseudaminobacter salicylatoxidans BN12 forms a peculiar gentisate 1,2-dioxygenase (SDO) that oxidatively cleaves gentisate (2,5-dihydroxybenzoate) and additionally 1-hydroxy-2-naphthoate, salicylate and various amino-, chloro-, fluoro-, hydroxy- and methylsalicylates. In the present study, the conversion of 5-fluorosalicylate by this enzyme was analysed using various analytical techniques. Spectrophotometric assays showed that the conversion of 5-fluorosalicylate by the purified enzyme resulted in the formation of a new unstable intermediate showing an absorbance maximum at λmax = 292 nm. The analysis of the enzymatic reaction by HPLC showed that two main products with absorbance maxima at λmax = 292–296 nm were formed from 5-fluorosalicylate. The same two products (although in different relative proportions) were also formed when the SDO transformed 5-chlorosalicylate or when a purified 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp. JS329 oxidized 5-nitrosalicylate. A whole cell system with recombinant Escherichia coli cells overexpressing the SDO activity was established in order to produce larger amounts of the reaction products. The reaction products were subsequently identified by 1H-NMR and mass spectrometry as stereoisomers of 2-oxo-3-(5-oxofuran-2-ylidine)propanoic acid. The release of fluoride in the course of the dioxygenolytic cleavage reaction was confirmed by ion-chromatography and 19F-NMR. A novel reaction catalysed by a bacterial enzyme is described that splits with the help of oxygen the carbon–fluorine bond in non-natural compounds.