Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel
Chaloupkova, R., Sykorova, J., Prokop, Z., Jesenska, A., Monincova, M., Pavlova, M., Tsuda, M., Nagata, Y., Damborsky, J.
JOURNAL OF BIOLOGICAL CHEMISTRY 278: 52622-52628 (2003)
Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis: the residue partially blocks the entrance tunnel and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in fourteen proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, fifteen active protein variants were obtained suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterised by determination of specific activities with twelve halogenated substrates and steady-state kinetic parameters with two substrates. Effect of mutation on the enzyme activities varied dramatically with the structure of the substrates suggesting that extrapolation of one substrate to another may be misleading and a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and non-polar amino acid in the position 177. This result is consistent with the phylogenetic analysis which showed that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants. Although the approach did not result in mutant enzymes with entirely new capabilities it did result in improving the catalytic properties and expanding their substrate specificities.