| Description |
Haloalkane dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of haloalkane dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.
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