We report the biochemical characterization of a novel haloalkane dehalogenase DatA isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilising residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity towards racemic mixtures of chiral brominated alkanes and esters.
Haloalkane dehalogenases (HLDs; EC 18.104.22.168) are enzymes that catalyse the dehalogenation of alkyl halides, and are found in a wide range of bacteria (1). They promote the hydrolysis of a broad range of halogenated compounds, cleaving the carbon-halogen bond to generate the corresponding alcohol, a halide and a proton (3). A number of halogenated compounds are environmentally toxic industrial byproducts, and it has been suggested that haloalkane dehalogenases may be useful catalysts for their biodegradation, with potential applications in bioremediation. In biocatalysis, there is a long-standing interest for these enzymes particularly for the production of optically pure alcohols. Therefore, the identification of dehalogenating enzymes with appropriate selectivity patterns is very important in terms of their industrial utility (3-4, 12-14).
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Agrobacterium tumefaciens causes the plant disease crown gall by transferring a discrete set of genes located on the tumor inducing (Ti) plasmid into the plant's cells. These genes are then integrated and expressed, resulting in the disease (15). The open reading frame atu6064 (AE009425), which we refer to as datA, is located on the Ti plasmid of Agrobacterium tumefaciens C58. A recent phylogenetic analysis suggested that the translation product DatA from Agrobacterium tumefaciens C58 is a putative member of HLD subfamily II. HLDs of this subfamily have a characteristic catalytic pentad composed of Asp-His-Glu and Asn-Trp residues (1). Sequence alignment revealed that DatA differs from all of the other known HLDs in that one of its halide stabilizing residues is a tyrosine rather than the tryptophan observed in other members of the family. The halide-stabilizing residues play a key role in the binding of a halogenated substrate and in the stabilization of the transition state of carbon-halogen bond-breaking transition (3).
The start codon of datA has been predicted differently by the two groups who determined independently the entire genome sequence of C58. Therefore, we predicted another start codon of datA on the basis of comparison with other HLDs and existence of the most probable SD sequence upstream of our predicted start codon. An optimized synthetic gene coding for DatA with a six-histidine tag on the C-terminus was expressed under the tac promoter in Escherichia coli strain Arctic Express (Stratagene, La Jolla, USA). The DatA so produced was purified to homogeneity by Q-Sepharose ion exchange chromatography (GE Healthcare, Waukesha, USA) and affinity chromatography with Ni-nitrilotriacetic acid resin (Qiagen, Hilden, Germany), with a yield of 30 mg of purified protein per litre of culture.
Circular dichroism spectroscopy in the far-UV region was used to assess the folding and stability of DatA. The spectrum of pure DatA contains two negative peaks at 208 and 222 nm and one positive peak at 195 nm (Fig. 1) and is similar to other characterized HLDs possessing ï¡/ï¢-hydrolase fold (6-7, 10-11, 13). The differences between the depths of the minima for DatA are due to differences in the abundance of its helical structures. The thermal stability of DatA was measured by heating a solution of the protein from 22 to 80°C at rate 1°C min-1 and monitoring the changes in its ellipticity at 222 nm. The melting temperature of DatA was found to be 48.3 ± 0.2°C, which is comparable to the melting temperatures of other HLDs (6-7, 10). 1,3-dibromopropane was used to examine the effect of temperature and pH on the activity of DatA; peak activity was observed at 40oC and pH 9.8. These values are consistent with those observed with other HLDs (5-10, 14).
The native structure of DatA was examined by gel filtration with a Sephacryl S-500 HR (GE Healthcare, Waukesha, USA), using an elution buffer consisting of 50 mM Tris-HCl at pH 7.5 in the presence of 150 mM of NaCl, and by native electrophoresis using the same buffer with or without 150 mM of NaCl. DatA exists as a monomer with an estimated molecular mass of 34 kDa under high salt conditions (Fig. 2 and Fig. 3A), but is dimeric under low salt conditions (Fig. 3B). This suggests that ionic interactions are involved in the (dis)association of the protein.
Figure 2 and Figure 3
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The specificity of DatA was studied using 36 different halogenated substrates. It was found to be highly active towards brominated and iodinated compounds, but poorly active towards most of the chlorinated compounds. The highest activity (0.023 ïmol s-1 mg-1 of protein) was observed with 1,3-dibromopropane (Fig. 4).
A detailed kinetic analysis was performed with six selected substrates, consisting of brominated, chlorinated and iodinated analogs of 1-halohexane and 1,3-dihalopropane. Kinetics of DatA with 1,3-dibromopropane (K0.5 = 2.15 ± 0.15 mM; kcat = 1.47 ± 0.08 s-1) and 1-bromohexane (K0.5 = 0.14 ± 0.09 mM; kcat = 1.7 ± 0.1 s-1) follow single hyperbolic relationship with Hill coefficient (nH value) equal to 1. Interestingly, sigmoidal kinetics were observed with 1,3-diiodopropane and 1-chlorohexane, suggesting that these species react with DatA via a mechanism involving cooperative substrate binding. In keeping with this suggestion, Hill coefficients (nH values) of 3.5 ± 1.6 and 1.27 ± 0.23, respectively, were observed with these two substrates. Other unusual kinetics of DatA is a combination of cooperative mechanism with substrate inhibition observed in the case of 1-iodohexane with K0.5 was 0.63 ± 0.25 and substrate inhibition constant (Ksi) was 1.7 ± 0.1 mM. The Hill-coefficients nH and mH for this substrate were 2.4 ± 1.0 and 11.6 ± 6.0, respectively (Table 1). However, the kinetics of the DatA with 1,3-dichloropropane could not be accurately measured. The reaction velocity of DatA towards this substrate increased linearly between 0 and 4 mM, indicating that the K0.5 was significantly higher than the solubility limit (4 mM) of the substrate (Table 1). Taken together, these results indicate that the kinetic mechanism by which DatA achieves haloalkane hydrolysis is strongly substrate-dependent.
The enantioselectivity of DatA was assessed by determining the kinetics of hydrolysis of racemic brominated alkanes and esters. These data make it possible to quantify the ability of the dehalogenase to discriminate between two enantiomers present at equal concentrations (12-13). DatA exhibited a high ability to discriminate between the R- and S- enantiomers of 2-bromopentane, 2-bromohexane, and ethyl 2-bromopropionate (E > 200). Low to medium selectivities were observed with 2-bromobutane (E = 6) and methyl 2-bromopropionate (E = 54), while no activity was detected with methyl and ethyl 2-bromobutyrate (Table 2). To the best of our knowledge, DatA is the most highly enantioselective of all the HLDs reported to date towards bromoalkane substrates, and thus may be of considerable interest in biocatalysis (3, 12-13).
Nucleotide sequence accession number. The nucleotide sequence of the datA gene was deposited in the EMBL/GenBank/DDBJ database under accession number AB478945.
This work was supported by The Ministry of Education, Youth, and Sports of the Czech Republic (LC06010 to T.K., MSM0021622412 to Z.P., and CZ.1.05/2.1.00/01.0001 to R.C.), the Grant Agency of the Czech Academy of Sciences (IAA401630901 to J.D.) and Grant-in-Aids from The Ministry of Education, Culture, Sports, Science, and Technology and The Ministry of Agriculture, Forestry, and Fisheries, Japan (to Y.N.).