Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB

Abstract

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.

FIG. 1.

Alignment of amino acid sequences of LinB and Rv2579. The secondary structure elements (indicated by lines under the sequence), the catalytic triad (indicated by triangles above the sequence), and the active site and tunnel residues (indicated by boxes) of LinB were deduced from the crystal structure (17). The active site residues that are different in LinB and Rv2579 are shaded.

FIG. 2.

Stereo picture of the three-dimensional structure of haloalkane dehalogenase LinB, with the mutated residues indicated. The protein structure is represented by the alpha trace; the substrate molecule 1-chlorobutane (colored by atom type) docked to the active site and mutated amino acid residues (in red) are represented by sticks.

FIG.3.

(A) Scatter plot of the first score (t1) plotted against the second score (t2). (B and C) Column plots of the first loading p1 (B) and the second loading p2 (C). The scores and loadings were obtained from the principal component analysis applied to the 6 × 34 (mutants × substrates) data matrix. The first principal component explained 70% of the data variance, and the second principal component explained 12% of the data variance.