Structural and biochemical characterization of novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94 revealed two halide binding sites in haloalkane dehalogenases

T. Prudnikova¹, T. Mozga², P. Řezáčová^3,4, R. Chaloupková², Y. Sato⁵, M. Kutý^1,6, Z. Prokop², Y. Nagata⁵, J. Damborský², I. Kutá Smatanová^1,6

¹Institute of Physical Biology, University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady

²Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno

³Institute of Systems Biology and Ecology, Academy of Science of Czech Republic, Zamek 136, 373 33 Nove Hrady

⁴Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague

⁵Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

⁶Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague

jiri@chemi.muni.cz

Keywords: haloalkane dehalogenases, structure-function relationships, two halide binding sites

A novel haloalkane dehalogenase DbeA belonging to the subfamily HLD-II [1] was isolated from soil bacteria Bradyrhizobium elkani USDA94. This new enzyme is closely related to DbjA from Bradyrhizobium japonicum USDA110 [2]. Proper folding of DbeA was assessed by measurement of CD spectra in far-UV and near-UV spectral regions. Thermal stability of DbeA was evaluated by determination of the melting temperature (Tm = 58.5 ± 0.2°C), which is in the similar range as structure stability observed for other family members. Molecular weight determined by gel filtration and native polyacrylamide gel electrophoresis confirmed dimeric state of DbeA under native conditions. Activity data of HLDs were measured with a set of 30 various substrates. The principal component analysis of the specific activities showed that DbeA is less active than DbjA and posses a unique substrate specificity. This enzyme has the highest activity towards brominated and iodinated compounds from all tested HLDs. DbeA showed high enantioselective conversion of 2-bromopentane, 2-bromohexane and brominated ester of propionic and butyric acid into chiral alcohols. The temperature and pH profiles of DbeA were detected by activity measurement with 1-iodohexane as a substrate. The highest activity of the enzyme was detected at the temperature range 45-55°C, which is in a good agreement with the temperature profiles of other HLDs. Surprisingly, DbeA showed more than one pH optimum with the maximal activity detected at pH conditions 6.0 and 8.5-9.5. Two pH optima were described only for DmbA, while other HLDs exhibited single pH optimum.

Crystallographic analysis of DbeA revealed the presence of two halide binding sites for chloride anion. The first chloride anion in DbeA structure was found in product-binding site where interacts with conserved halide binding residues Asn38 and Trp104. This binding site is common for all HLDs-II. The second chloride anion in DbeA structure is placed about 10 Å far from the product-binding site, buried deep in the protein core, where is coordinated by side chains of Gly37, Thr40, Ile44, Gln102 and Gln274. This chloride-binding site is unique to DbeA and its closely related enzyme DbjA. The full occupancy of this second chloride binding site and its location in close proximity of the active site suggests that this halide-binding site might have some biological relevance, perhaps on DbeA activity. To elucidate the role of the second halide binding site on DbeA structure and function, the two point mutant variant lacking the second binding site, DbeA I44L and Q102H, was constructed and characterized. The comparison of the wild type and mutant enzymes will be presented and discussed.

References

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2. Y. Sato, M. Monincova, R. Chaloupkova, Z. Prokop, Y. Ohtsubo, K. Minamisawa, M. Tsuda, J. Damborsky, Y. Nagata, Appl. Environ. Microbiol., 71, (2005), 4372.