Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and Institute of Complex Systems University of South Bohemia Zamek 136 37333 Nove Hrady Czech Republic

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Bergfors, T. M. (1999). Protein Crystallization International University Line: La Jolla, CA ,USA.

Bidmanova, S., Chaloupkova, R., Damborsky, J. & Prokop, Z. (2010). Anal. Bioanal. Chem. 398, 1891–1898. PubMed

Biedermannova, L., Prokop, Z., Gora, A., Chovancova, E., Kovacs, M., Damborsky, J. & Wade, R. C. (2012). Biol. Chem. 287, 29062–29074. PubMed PMC

Campbell, D. W., Müller, C. & Reardon, K. F. (2006). Biotechnol. Lett. 28, 883–887. PubMed

Chaloupkova, R., Prudnikova, T., Rezacova, P., Prokop, Z., Mozga, T., Koudelakova, T., Daniel, L., Brezovsky, J., Sato, Y., Kuty, M., Nagata, Y., Kuta-Smatanova, I. & Damborsky, J. (2013). In preparation.

Chaloupková, R., Sýkorová, J., Prokop, Z., Jesenská, A., Monincová, M., Pavlová, M., Tsuda, M., Nagata, Y. & Damborský, J. (2003). J. Biol. Chem. 278, 52622–52628. PubMed

Diederichs, K. & Karplus, P. A. (1997). Nature Struct. Biol. 4, 269–275. PubMed

Janssen, D. B., Dinkla, I. J. T., Poelarends, G. J. & Terpstra, P. (2005). Environ. Microbiol. 7, 1868–1882. PubMed

Kabsch, W. (2010). Acta Cryst. D66, 125–132. PubMed PMC

Koudelakova, T., Chovancova, E., Brezovsky, J., Monincova, M., Fortova, A., Jarkovsky, J. & Damborsky, J. (2011). Biochem. J. 435, 345–354. PubMed

Krug, M., Weiss, M. S., Heinemann, U. & Mueller, U. (2012). J. Appl. Cryst. 45, 568–572.

Los, G. V. & Wood, K. (2007). Methods Mol. Biol. 356, 195–208. PubMed

Marek, J., Vévodová, J., Smatanová, I. K., Nagata, Y., Svensson, L. A., Newman, J., Takagi, M. & Damborský, J. (2000). Biochemistry, 39, 14082–14086. PubMed

Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. PubMed

Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. (2006). Acta Cryst. D62, 859–866. PubMed

Mueller, U., Darowski, N., Fuchs, M. R., Förster, R., Hellmig, M., Paithankar, K. S., Pühringer, S., Steffien, M., Zocher, G. & Weiss, M. S. (2012). J. Synchrotron Rad. 19, 442–449. PubMed PMC

Nagata, Y., Miyauchi, K., Damborsky, J., Manova, K., Ansorgova, A. & Takagi, M. (1997). Appl. Environ. Microbiol. 63, 3707–3710. PubMed PMC

Ohana, R. F., Encell, L. P., Zhao, K., Simpson, D., Slater, M. R., Urh, M. & Wood, K. V. (2009). Protein Expr. Purif. 68, 110–120. PubMed

Prokop, Z., Sato, Y., Brezovsky, J., Mozga, T., Chaloupkova, R., Koudelakova, T., Jerabek, P., Stepankova, V., Natsume, R., van Leeuwen, J., Janssen, D., Florian, J., Nagata, Y., Senda, T. & Damborsky, J. (2010). Angew. Chem. Int. Ed. 49, 6111–6115. PubMed

Smatanová, I., Nagata, Y., Svensson, L. A., Takagi, M. & Marek, J. (1999). Acta Cryst. D55, 1231–1233. PubMed

Stucki, G. & Thueer, M. (1995). Environ. Sci. Technol. 29, 2339–2345. PubMed

Weiss, M. S. & Hilgenfeld, R. (1997). J. Appl. Cryst. 30, 203–205.

Weiss, M. S., Metzner, H. J. & Hilgenfeld, R. (1998). FEBS Lett. 243, 291–296. PubMed

Westerbeek, A., Szymański, W., Feringa, B. L. & Janssen, D. B. (2011). ACS Catal. 1, 1654–1660.

Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. PubMed

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

Crystallographic analysis of new psychrophilic haloalkane dehalogenases: DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17