Abstract The discovery of new enzymes that can hydrolyse the carbon–fluorine bond is important owing to the number of anthropogenic fluorinated compounds employed in a diverse range of applications and their recalcitrance in the environment. An enzyme from Serratia liquefaciens has been discovered that has dehalogenase, esterase and N -deformylase activities, a combination that has not previously been reported. The gene coding for the enzyme was expressed in Escherichia coli and the His-tagged protein purified via immobilised metal affinity chromatography. The enzyme defluorinated monofluoroethylglycine and fluoroacetate yielding homoserine and glycolate as defluorinated products, respectively. Esterase activity was established via p -nitrophenol acetate and p -nitrophenol butyrate hydrolysis; N -deformylation was measured by incubating the enzyme with N -formylmaleamic acid and detecting maleamic acid using gas chromatography-mass spectrometry. Based on sequence alignment, the catalytic triad was Ser-His-Asp, which is most unexpected as other fluoroacetate dehalogenases have a Asp-His-Asp triad. Site-directed mutagenesis of the presumed active site serine residue at position 101 to alanine resulted in the elimination of dehalogenating and esterase activities. In contrast, the S101D mutant had improved dehalogenase activity and diminished esterase activity compared to the wild type. Computational assessment of the presumptive catalytic triad (Ser-His-Asp) revealed a different orientation of His245 compared with the corresponding residue in fluoroacetate dehalogenase from Burkholderia sp. FA1. However, in the S101D mutant, the residues were much more closely aligned with those of the FA1 dehalogenase. Similar genes are present in other Serratia species and a number of other bacteria; thus, it is possible that other enzymes exist in the environment that have similar defluorinating activity. Such enzymes are a valuable resource for the development of biological methods for the remediation of organofluorine-polluted environments. Key points • SlDefH hydrolyses fluorinated substrates, esters and N-formylmaleamic acid • An S101D mutant has improved dehalogenase and reduced esterase activity • Enzymes with similar properties are likely to be present in the environment
Khan et al. (Mon,) studied this question.