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

Moriuchi R, Tanaka H, Nikawadori Y, Ishitsuka M, Ito M, Ohtsubo Y, Tsuda M, Damborsky J, Prokop Z, Nagata Y - AMB Express (2014)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan ; The United Graduate School of Agricultural Science, Gifu University 1-1 Yanagido, Gifu 501-1193, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

The β-HCH degradation activities of LinBUT, LinBMI, and their intermediate mutants. Specificity constants of LinBUT (vertical hexagon), LinBMI (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 LinBUT to LinBMI by the accumulation of seven point mutations is shown by arrows (a).
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Figure 5: The β-HCH degradation activities of LinBUT, LinBMI, and their intermediate mutants. Specificity constants of LinBUT (vertical hexagon), LinBMI (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 LinBUT to LinBMI by the accumulation of seven point mutations is shown by arrows (a).

Mentions: Since M2-1 showed only a weak LinBMI-type activity in the previous study (Ito et al. [2007]), we introduced further mutations into M2-1 in this study. However, for the critical comparison with other mutants, the seven single-point mutants [A81T (M1-1), A112V (M1-2), I134V (M1-3), A135T (M1-4), I138L (M1-5), A247H (M1-6), and M253I (M1-7)] of LinBUT and M2-1 were also kinetically characterized in this study, and the importance of individual mutations for LinBMI activity towards β-HCH was assessed (Figure 3 and Table 1). Among the seven single-point mutations, only the A112V mutation had a negative effect on β-HCH degradation activity, while the other six mutations showed a slightly positive effect on enzymatic activity towards β-HCH (Figure 3b-h and Figure 5). Interestingly, the A81T mutation had a relatively strong effect on the first conversion (β-HCH to PCHL) step (Figure 3b and Figure 5e). The involvement of T81 in the first step was consistent with the decrease in this step by the reciprocal T81A mutation into LinBMI (M6-1) (Figure 5e and Additional file 1: Figure S1b) (Okai et al. [2013]). M2-1 showed higher activity for the second conversion (PCHL to TCDL) step (Table 1) than all the single mutants, but its activity was still weak (Figure 3i and Figure 5a).


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

Moriuchi R, Tanaka H, Nikawadori Y, Ishitsuka M, Ito M, Ohtsubo Y, Tsuda M, Damborsky J, Prokop Z, Nagata Y - AMB Express (2014)

The β-HCH degradation activities of LinBUT, LinBMI, and their intermediate mutants. Specificity constants of LinBUT (vertical hexagon), LinBMI (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 LinBUT to LinBMI by the accumulation of seven point mutations is shown by arrows (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230811&req=5

Figure 5: The β-HCH degradation activities of LinBUT, LinBMI, and their intermediate mutants. Specificity constants of LinBUT (vertical hexagon), LinBMI (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 LinBUT to LinBMI by the accumulation of seven point mutations is shown by arrows (a).
Mentions: Since M2-1 showed only a weak LinBMI-type activity in the previous study (Ito et al. [2007]), we introduced further mutations into M2-1 in this study. However, for the critical comparison with other mutants, the seven single-point mutants [A81T (M1-1), A112V (M1-2), I134V (M1-3), A135T (M1-4), I138L (M1-5), A247H (M1-6), and M253I (M1-7)] of LinBUT and M2-1 were also kinetically characterized in this study, and the importance of individual mutations for LinBMI activity towards β-HCH was assessed (Figure 3 and Table 1). Among the seven single-point mutations, only the A112V mutation had a negative effect on β-HCH degradation activity, while the other six mutations showed a slightly positive effect on enzymatic activity towards β-HCH (Figure 3b-h and Figure 5). Interestingly, the A81T mutation had a relatively strong effect on the first conversion (β-HCH to PCHL) step (Figure 3b and Figure 5e). The involvement of T81 in the first step was consistent with the decrease in this step by the reciprocal T81A mutation into LinBMI (M6-1) (Figure 5e and Additional file 1: Figure S1b) (Okai et al. [2013]). M2-1 showed higher activity for the second conversion (PCHL to TCDL) step (Table 1) than all the single mutants, but its activity was still weak (Figure 3i and Figure 5a).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan ; The United Graduate School of Agricultural Science, Gifu University 1-1 Yanagido, Gifu 501-1193, Japan.

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.

No MeSH data available.


Related in: MedlinePlus