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 14 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, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The 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 that 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 nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing 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 sitedirected 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.Haloalkane dehalogenases are microbial enzymes acting on haloorganic compounds. The enzymes cleaves the carbon-halogen bond and replaces a halogen with a hydroxyl group from a water molecule (1). Activity and specificity of haloalkane dehalogenases is not optimal for industrial applications (2), and numerous studies have been conducted to improve their catalytic properties using in vitro techniques (3-13). Engineered enzymes can be used in biotechnology applications, such as detoxification of environmental pollutants and bioorganic synthesis. Such technologies are already in use (14) or are under development (2, 15, 16). Furthermore, haloalkane dehalogenases has become an important model system for in silico study of molecular principles of enzymatic catalysis (12,(17)(18)(19)(20)(21)(22)(23)(24).Haloalkane dehalogenase LinB (25) is the enzyme isolated from a ␥-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimob...

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The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.