Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2

Abstract

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-l-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50°C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.Both epichlorohydrin (3-chloro-1,2-epoxypropane) and its precursor in chemical synthesis, 1,3-dichloro-2-propanol, are industrial chemicals that may enter the environment because of their volatile character or improper disposal. These compounds are considered important environmental pollutants (12), in part because epichlorohydrin has been shown to be mutagenic and carcinogenic in rats (18).Recently, three bacterial cultures able to grow on epichlorohydrin, 3-chloro-1,2-propanediol, or 1,3-dichloro-2-propanol were isolated (20). Two of these cultures, Pseudomonas sp. strain AD1 and Arthrobacter sp. strain AD2, were examined in more detail. Degradation of epichlorohydrin proceeded via 3-chloro-1,2-propanediol and glycidol (3-hydroxy-1,2-epoxypropane). Arthrobacter sp. strain AD2 was not capable of enzymatic opening of the epoxide ring in epichlorohydrin, but Pseudomonas sp. strain AD1 possessed an epoxide hydrolase, producing 3-chloro-1,2-propanediol., It was shown that both strains dehalogenated haloalcohols by a highly ihducible enzyme which we call haloalcohol dehalogenase (20). The dehalogenase catalyzed the conversion of vicinal haloalcohols to the corresponding epoxides with liberation of inorganic chloride or bromide. The same mechanism of dehalogenation of haloalcohols was found earlier in a Flavobacterium sp. isolated on 2,3-dibromo-1-propanol (1, 3).The products of dehalogenation and the substrate range of the enzyme suggest that haloalcohol dehalogenases do not belong to other established classes of dehalogenases, such as haloacid dehalogenases (4, 15), haloalkane dehalogenases (6-8, 10, 16, 22), and dichloromethane dehalogenases (11,17). Since no information on the biochemical characteristics of haloalcohol dehalogenases is available, we purified and studied the enzyme from Arthrobacter sp. strain AD2 in more detail. We also present evidence that the dehalogenase of Pseudomonas sp. strain AD1 differs from the...

Genetics and Biochemistry of Dehalogenating Enzymes

“…Earlier, our group reported the presence of a similar enzyme in Arthrobacter sp. strain AD2 (74). This enzyme appeared to be a dimeric protein and has an N-terminal amino acid sequence similar to the Corynebacterium enzyme.…”

“…Haloalcohol lyases, also called halohydrin hydrogen halide lyases (41,74), catalyze the intramolecular nucleophilic displacement of vic-haloalcohols to their corresponding epoxides. The reaction has been studied for several halowww.annualreviews.org/aronline Annual Reviews Figure 10 Microbial dehalogenation of halopropanols via haloepoxides.…”

Isolation and Characterization of a 1,3-Dichloro-2-Propanol-Degrading Bacterium

“…The 1,3-DCP-dechlorinating enzyme of strain PY1 designated as Deh-PY1 was observed in the cytoplasmic fraction, similar to various haloalcohol-dehalogenases reported in previous papers. 6,8,9,21) Purified Deh-PY1 having a molecular mass of 80 kDa was composed of 4 identical 20 kDasubunits. The Km value and Vmax of Deh-PY1 were 2.67 mM and 7.81 µmol/min/mg, respectively, and the optimum reaction temperature and pH were 40-50°C and 9.5-10.5, respectively.…”

“…Enzymatic characteristics and activity against various haloalcohols of Deh-PY1 are compared with enzymes reported by other authors in Tables 3 and 4. Deh-PY1 was obtained from a bacterium of the genus Arthrobacter similar to AD2 9,22) and DehA, 6) but their characteristics differ. Deh-PY1 and AD2 are homo tetramer and dimer, respectively, whereas DehA is a hetero polymer.…”

“…Therefore, various studies have been made to isolate organisms with the ability to use such compounds in order to develop a process of eliminating the halogenated pollutants. Microorganisms dechlorinating 1,3-DCP have also been [6][7][8][9] However, information on 1,3-DCP-degtrading organisms available for use in a biological control system remains inadequate.…”