Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards β-hexachlorocyclohexane

Abstract

Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards β-hexachlorocyclohexane (β-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's β-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.

Keywords: Biodegradation; Haloalkane dehalogenase; Protein evolution; Xenobiotics; β-Hexachlorocyclohexane.

Figure 1

β-HCH degradation reactions catalyzed by LinB_UTand LinB_MI. LinB_MI converts β-HCH to PCHL and further to TCDL, while LinB_UT catalyzes only the first conversion step of β-HCH to PCHL.

Figure 2

Structure of LinB_MI (PDB code 4H77) (Okai et al. [2013]) and location of catalytic triad (D108, E132, and H272; shown in red) and the seven dissimilar amino acid residues between LinB_MI and LinB_UT: V134 and V112 (in magenta), L138, H247, and I253 (in cyan), T135 (in green), and T81 (in blue). See text for detail.

Figure 3

Degradation of β-HCH in reaction mixtures containing LinB_UTand its mutant derivatives. LinB_UT wild-type (a) and its mutants, M1-1 (b), M1-2 (c), M1-3 (d), M1-4 (e), M1-5 (f), M1-6 (g), M1-7 (h), M2-1 (i), M3-1 (j), M3-2 (k), M3-3 (l). The closed circle and closed and open triangles represent β-HCH, PCHL, and TCDL, respectively. Each value given is the mean of triplicates. Kinetic data were fitted to the irreversible two-step reaction scheme of β-HCH conversion to TCDL via PCHL (Scheme 1 in Materials and methods) by using GEPASI 3.2 software (Mendes [1997]) and shown in solid lines. The specificity constants and their standard errors for both reaction steps (k₁ and k₂) were obtained from the calculation (Table 1).

Figure 4

Degradation of β-HCH in reaction mixtures containing LinB_UTmutant derivatives. M4-1 (m), M4-2 (n), M5-1 (o), M5-2 (p), M5-3 (q), M5-4 (r), and M5-5 (s). See legend of Figure 3.

Figure 5

The β-HCH degradation activities of LinB_UT, LinB_MI, and their intermediate mutants. Specificity constants of LinB_UT (vertical hexagon), LinB_MI (circle), and their intermediate mutants (single, open triangle; double, horizontal hexagon; 3-point, pentagon; 4-point, square; 5-point, diamond; and 6-point, closed triangle) for the first conversion (from β-HCH to PCHL: X axis) and the second conversion (from PCHL to TCDL: Y axis) steps (Table 1) were plotted in logarithmical values. The effects of A112V (b), I138L (c), M253I (d), A81T (e), and A135T (f) mutations were extracted from the total plot (a) and shown by arrows. One potential evolutionary route from LinB_UT to LinB_MI by the accumulation of seven point mutations is shown by arrows (a).