This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes
drbA from
Rhodopirellula baltica SH1 and
dmbC from
Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.Haloalkane dehalogenases (EC 3.8.1.5.) acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (
7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (
4,
6,
22), construction of biosensors (
2), decontamination of warfare agents (
17), and synthesis of optically pure compounds (
19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (
3). In contrast to subfamilies HLD-I and HLD-II, the subfamily HLD-III is currently lacking experimentally characterized proteins. We have therefore focused on the isolation and study of two selected representatives of the HLD-III subfamily, DrbA and DmbC.The
drbA gene was amplified by PCR using the cosmid pircos.a3g10 originating from marine bacterium
Rhodopirellula baltica SH1, and the
dmbC gene was amplified from DNA originating from obligatory pathogen
Mycobacterium bovis 5033/66. Six-histidine tails were added to the C termini of DrbA and DmbC in a cloning step, enabling single-step purification using Ni-nitrilotriacetic acid resin. Haloalkane dehalogenase DrbA was expressed under the T7 promoter and purified, with a resulting yield of 0.1 mg of protein per gram of cell mass. Haloalkane dehalogenase DmbC was obtained by expression in
Mycobacterium smegmatis, with a yield of 0.07 mg of purified protein per gram of cell mass.The correct folding and secondary structures of the newly prepared enzymes were verified by circular dichroism (CD) spectroscopy. Far-UV CD spectra were recorded for DrbA and DmbC enzymes and other, related haloalkane dehalogenases. All enzymes tested exhibited CD spectra with two negative features at 208 and 222 nm and one positive peak at 195 nm, which are characteristic of α-helical content (Fig. ). This suggested that both new enzymes, DrbA and DmbC, were folded correctly. However, DmbC exhibited more intense negative maxima which differed from other haloalkane dehalogenases in the θ
222/θ
208 ratio. This finding indicated a slight variation in the arrangement of secondary structure elements of the DmbC enzyme. Thermally induced denaturations of DrbA and DmbC were tested in parallel. Both enzymes showed changes in ellipticity during increasing temperature. The melting temperatures calculated from these curves were 45.8 ± 0.4°C for DmbC and 39.4 ± 0.1°C for DrbA. The thermostability results obtained for DrbA and DmbC were in good agreement with the range of melting temperatures determined for other, related haloalkane dehalogenases.
Open in a separate windowFar-UV CD spectra of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Protein concentration used for far-UV CD spectrum measurement was 0.2 mg/ml.The sizes of the purified proteins were estimated by electrophoresis under native conditions conducted using a 10% polyacrylamide gel (Fig. ). More precise determination of the sizes of DrbA and DmbC was achieved by gel filtration chromatography performed on Sephacryl S-500 HR (GE Healthcare, Uppsala, Sweden), calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (Fig. ). Both DrbA and DmbC were eluted from the column in the fraction prior to blue dextran, indicating that both enzymes form oligomeric complexes of a size larger than 2,000 kDa (Fig. ). The haloalkane dehalogenases which have been biochemically characterized so far form monomers, except for DbjA isolated from
Bradyrhizobium japonicum USDA110 (
21), which shows monomeric, dimeric, and tetrameric forms according to the pH of the buffer (R. Chaloupkova, submitted for publication).
Open in a separate windowNative protein electrophoresis of DrbA and DmbC. Lane 1, carbonic anhydrase (29 kDa); lane 2, ovalbumin (43 kDa); lane 3, bovine albumin (67 kDa); lane 4, conalbumin (75 kDa); lane 5, catalase (240 kDa); lane 6, ferritin (440 kDa); lane 7, DrbA; lane 8, DmbC.
Open in a separate windowGel filtration chromatogram of DrbA and DmbC. (A) The following calibration kit samples (0.5 ml of a concentration of 2 mg/ml protein loaded) were analyzed using 50 mM Tris-HCl with 150 mM NaCl, pH 7.5, as elution buffer: blue dextran (line 1, 9.6-ml fraction), thyroglobulin (line 2, 15.95-ml fraction), ferritin (line 3, 16.78-ml fraction), ovalbumin (line 4, 18.55-ml fraction), and RNase A (line 5, 20.08-ml fraction). (B and C) Haloalkane dehalogenase DrbA eluted in the 9.03-ml fraction (B), and haloalkane dehalogenase DmbC in the 9.31-ml fraction (C).The substrate specificities of DrbA and DmbC were investigated with a set of 30 selected chlorinated, brominated, and iodinated hydrocarbons. Standardized specific activities related to 1-chlorobutane (summarized in Table ) were compared with the activity profiles of other haloalkane dehalogenases (Fig. ). DrbA and DmbC displayed similar activity patterns, with catalytic activities approximately two orders of magnitude lower than those of other known haloalkane dehalogenases (
1,
5,
8-
11,
13-
16,
18,
20,
23). HLD-III subfamily enzymes showed a restricted specificity range and a preference for iodinated short-chain hydrocarbons. Both phenomena may be related to the composition of the catalytic pentad Asp-His-Asp+Asn-Trp, which is unique to the members of the HLD-III subfamily (
3). The preference for substrates carrying an iodine substituent can be related to a pair of halide-binding residues and their spatial arrangement with the catalytic triad. These residues make up the catalytic pentad, playing a critical role in substrate binding, formation of the transition states, and the reaction intermediates of the dehalogenation reaction (
12).
Open in a separate windowSubstrate specificity profiles of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Activities were determined using a consistent set of 30 halogenated substrates (see Table ). Data were standardized by dividing each value by the sum of all activities determined for individual enzymes in order to mask the differences in absolute activities. Specific activities (in μmol·s
−1·mg
−1) with 1-chlorobutane are 0.0003 (DrbA), 0.0001 (DmbC), 0.0003 (DatA), 0.0133 (DbjA), 0.0010 (DbeA), 0.0128 (DhaA), 0.0231 (LinB), 0.0171 (DmbA), and 0.0117 (DhlA).
TABLE 1.
Specific activities of haloalkane dehalogenases DrbA and DmbC toward a set of 30 halogenated hydrocarbons
aSubstrate | DrbA
| DmbC
|
---|
Sp act (nmol product·s−1· mg−1 protein) | Relative activity (%) | Sp act (nmol product·s−1· mg−1 protein) | Relative activity (%) |
---|
1-Chlorobutane | 0.291 | 100 | 0.122 | 100 |
1-Chlorohexane | 0.129 | 44 | 0.122 | 100 |
1-Bromobutane | 0.081 | 28 | 1.221 | 1,000 |
1-Bromohexane | 0.181 | 62 | 0.977 | 800 |
1-Iodopropane | 0.143 | 49 | 2.198 | 1,800 |
1-Iodobutane | 0.506 | 174 | 2.564 | 2,100 |
1-Iodohexane | 0.095 | 33 | 0.244 | 200 |
1,2-Dichloroethane | NA | NA | NA | NA |
1,3-Dichloropropane | NA | NA | 0.012 | 10 |
1,5-Dichloropentane | NA | NA | 0.061 | 50 |
1,2-Dibromoethane | 0.098 | 34 | 0.855 | 700 |
1,3-Dibromopropane | NA | NA | 5.007 | 4,100 |
1-Bromo-3-chloropropane | 0.001 | 0 | 1.465 | 1,200 |
1,3-Diiodopropane | 0.358 | 123 | 6.716 | 5,500 |
2-Iodobutane | 0.028 | 9 | NA | NA |
1,2-Dichloropropane | NA | NA | NA | NA |
1,2-Dibromopropane | 0.148 | 51 | 0.244 | 200 |
2-Bromo-1-chloropropane | 0.091 | 31 | 0.488 | 400 |
1,2,3-Trichloropropane | NA | NA | NA | NA |
Bis-(2-chloroethyl) ether | NA | NA | NA | NA |
Chlorocyclohexane | NA | NA | NA | NA |
Bromocyclohexane | 0.026 | 9 | NA | NA |
(1-Bromomethyl)-cyclohexane | NA | NA | 0.089 | 73 |
1-Bromo-2-chloroethane | 0.167 | 57 | 0.111 | 91 |
Chlorocyclopentane | NA | NA | NA | NA |
4-Bromobutyronitrile | 0.200 | 69 | 0.444 | 364 |
1,2,3-Tribromopropane | NA | NA | 0.222 | 182 |
3-Chloro-2-methyl propene | NA | NA | NA | NA |
2,3-Dichloropropene | 0.276 | 95 | NA | NA |
1,2-Dibromo-3-chloropropane | 0.010 | 3 | 0.044 | 36 |
Open in a separate windowaNA, no activity detected.Substrates 1-iodobutane and 1,3-diiodopropane, identified as the best substrates for haloalkane dehalogenases DrbA and DmbC, were used for measuring the dependency of enzyme activity on temperature and for determination of the pH optima. DrbA exhibited the highest activity with 1-iodobutane at 50°C, although above this temperature, the enzyme rapidly became inactivated. DmbC showed the highest activity toward 1,3-diiodopropane at 40°C, which is similar to the temperature determined with the haloalkane dehalogenases DmbA and DmbB (45°C), isolated from the same species (
10). Irrespective of the reaction temperature, DrbA showed the maximum activity at pH 9.15. DrbA kept 80% of its activity throughout a relatively wide range of pH values (pH 7.00 and 9.91) compared to DmbC, which showed a sharp maximum at pH 8.30. The Michaelis-Menten kinetics of DrbA and DmbC determined by isothermal titration microcalorimetry were investigated with 1-iodobutane, which is an iodinated analogue of 1-chlorobutane routinely used for characterization of haloalkane dehalogenases. The low magnitudes of the Michaelis constants (
Km = 0.063 ± 0.003 mM for DrbA and 0.018 ± 0.001 mM for DmbC) suggest a high affinity of both enzymes for 1-iodobutane. The catalytic constants determined with 1-iodobutane (
kcat = 0.128 ± 0.002 s
−1 for DrbA and 0.0715 ± 0.0004 s
−1 for DmbC) suggest that the low specific activities observed during substrate screening are not due to poor affinity but are instead due to a low conversion rate.The biochemical characteristics of purified DrbA and DmbC suggest that these proteins represent novel enzymes differing from previously characterized haloalkane dehalogenases by (i) their unique ability to form oligomers and (ii) low levels of dehalogenating activity with typical substrates of haloalkane dehalogenases. This study further illustrates how genome sequencing projects and phylogenetic analyses contribute to the identification of novel enzymes. Characterization of DrbA and DmbC, belonging to the subfamily HLD-III, partially filled a gap in the knowledge of the haloalkane dehalogenase family and provided an additional insight into evolutionary relationships among its members.
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