title: Biochemical characterization of a haloalcohol dehalogenase from Arthrobacter sp H10a
Creators: name: Serrado Assis, Helena Maria
Publisher: name: University of Kent
Year: 2021

description: Haloalcohols are an important class of industrial pollutants some of which are believed to be carcinogenic. Bacteria that are capable of efficient degradation of these compounds have been isolated. In our laboratory several soil isolates have been isolated by enrichment on 1,3-dichloro-2-propanol and have been subject of biochemical and physiological characterization. In order to shed more light on the biochemical and molecular mechanisms of dehalogenation an Arthrobaxter SP H10a was chosen for this study. Arthrobacter sp strain H10a possesses two enzymes capable of dehalogenating halohydrins (haloalcohol dehalogenases). These dehalogenases designated as Deh1 and Deh2 are expressed constitutively but levels of enzyme activity can be increased 3 to 4 fold in the presence of glycidol. The Deh1 enzyme showed higher activity towards 1,3-DCP while the Deh2 dehalogenase showed higher activity towards CPD. The analysis of the ratio of dehalogenation rate for both CPD and 1,3-DCP showed that addition of haloalcohols to the growth medium resulted in an increase in the Deh2 enzyme activity in relation to the Deh1 activity, whilst epoxides had an opposite effect. In an attempt to understand the mechanisms of dehalogenation the Deh1 haloalcohol dehalogenase was purified and characterized. The enzyme is constituted by two subunits of 31.5 and 34 kDa molecular weight, that associate with other proteins to form a large protein complex of 200 kDa. Peptide mapping with different proteases and amino acid micro-sequence analysis of tryptic digests showed 100% identity between the two Deh1 subunits. The Deh1 haloalcohol dehalogenase catalyzed the conversion of civinal halohydrins to epoxides and the reverse reaction in the presence of an excess of halogen. This enzyme showed maxium activity at 50°C and a broad pH optimum from 8.5 to 10.5. The apparent K/(_m/) and V\(_{max}\) values for dehalogenation of 1,3-DCP and CPD were 0.11mM, 236 μmol min\(^{-1}\) mg\(^{-1}\), respectively. The enzyme activity was inhibited by MCA and DCA. The inhibition pattern suggested a mixed type inhibition predominantly uncompetitive. Amino acid modifying experiments have shown that one of more cysteine and arginine residues may be involved in catalysis or play important roles in maintenance of the enzyme structure. Modification of histidine, lysine, aspartic and glumatic acids had no effect on the dehalogenase activity. Studies of the stereospecificity of the epoxides formed by the Deh1 haloalcohol dehalogenase revealed that (R)-ECH was selectively produced from 1,3-DCP. During the reverse reaction (R)-ECH was stereoselectively halogenated to 1,3-DCP if the halogen in the reaction mixture of chloride. However, if chloride was substituted by bromide, the (S)-isomer was halogenated preferentially. Although the enantiomeric excess and the yields of ECH obtained were low it was shown that it is possible to produce both isomers of ECH if the reaction conditions were optimized. An antibody raised against the Deh1 enzyme was used to screen other bacterial isolates in the laboratory culture collection. This antibody showed immunocross-reactivity with a haloalcohol dehalogenase from strain H10c. This enzyme revealed the same electrophoretic mobility as the Deh1 protein under both native and denaturing conditions. The Deh1 antibody also showed cross-reaction with a 31.5 kDa protein from strain H10f. No immunological cross-reactivity was found between this antibody and the total protein extracts from haloacid and haloalkane degrading bacteria.