MODIFICATION OF ACTIVITY, SPECIFICITY AND STABILITY OF HALOALKANE DEHALOGENASES BY ENGINEERING OF BURIED HALIDE-BINDING SITES

R. Chaloupkova¹, T. Prudnikova², P. Řezáčová^3,6, Z. Prokop¹, T. Koudeláková¹, L. Daniel¹, J. Brezovský¹, W. Ikeda-Ohtsubo⁴, Y. Sato⁴, M. Kutý^2,5,7, Y. Nagata⁴, I. Kutá Smatanová^2,5,7 and J. Damborský¹

¹Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic

²Institute of Complex Systems and CENAKVA, Faculty of Fisheries and Protection of Waters, University of South Bohemia, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic

⁴Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

⁵Institute of Nanobiology and Structural Biology GCRC, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

⁶Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, 166 10 Prague 6, Czech Republic

⁷Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

Haloalkane dehalogenases (HLDs; EC 3.8.1.5) are bacterial enzymes cleaving a carbon-halogen bond by a hydrolytic dehalogenation [1]. Crystal structure of the haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94 was solved to 2.2 Å resolution and revealed the presence of two binding sites for chloride aniont. The first chloride anion was found in the active site in between two conserved halide-stabilizing residues Asn38 and Trp104. This binding site is common for all members of the subfamily HLD-II [2]. The second chloride anion is placed about 10 Å from the first binding site, buried deep in the protein core, where it is coordinated by the side chains of Gln274, Gln102, Gly37 and Thr40. This second halide-binding site is unique to DbeA and its closely related enzyme DbjA [3,4] from Bradyrhizobium japonicum USDA110 (71% sequence identity) and has not been observed in any other crystal structure of HLD-II enzymes.

To elucidate the effect of the second halide-binding site on the structure and function of DbeA enzyme, two-point variant I44L+Q102H lacking the second halide-binding site was constructed, purified and biochemically characterized. Its comparison with the wild type enzyme revealed that elimination of the second halide-binding site decreased the stability of the enzyme in the presence of chloride salt and decreased its catalytic activity without change of its catalytic mechanism. Moreover, the two-point substitution resulted in a shift of the substrate-specificity class, which is the first time this has been demonstrated for the haloalkane dehalogenase enzyme family. Changes in the catalytic activity of the variant were attributed to deceleration of the rate-limiting hydrolytic step, mediated by lower basicity of the catalytic histidine. Our study demonstrates that engineering of the buried halide-binding sites has significant impact on enzyme’s activity, specificity and stability. Rational design of buried halide-binding sites could represent a novel strategy for engineering of biocatalysts with desired catalytic properties.

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3. Y. Sato, M. Monincová, R. Chaloupková, Z. Prokop, Y. Ohtsubo, K. Minamisawa, M. Tsuda, J. Damborský & Y. Nagata, Appl. Environ. Microbiol., 71 (2005) 4372-4379.

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