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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

Degradation of β-HCH in reaction mixtures containing LinBUTand its mutant derivatives. LinBUT 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).
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Figure 3: Degradation of β-HCH in reaction mixtures containing LinBUTand its mutant derivatives. LinBUT 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).

Mentions: In this study, cumulative mutations were introduced into LinBUT, and the resulting intermediate mutant enzymes between LinBUT and LinBMI were characterized in order to gain more insight into the molecular evolution of LinB towards β-HCH degradation activity. Since the LinBUT I134V/A247H (=M2-1) mutant showed only weak LinBMI-type activity in our previous study (Ito et al. [2007]), cumulative mutations at the positions A112, I138, and M253 were introduced into the M2-1 mutant. On the basis of kinetic analyses of the resulting three-, four-, and five-point LinBUT mutants (Figures 3, 4 and Table 1), enhancement of the catalytic performance of LinBUT towards β-HCH is discussed.


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)

Degradation of β-HCH in reaction mixtures containing LinBUTand its mutant derivatives. LinBUT 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Degradation of β-HCH in reaction mixtures containing LinBUTand its mutant derivatives. LinBUT 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).
Mentions: In this study, cumulative mutations were introduced into LinBUT, and the resulting intermediate mutant enzymes between LinBUT and LinBMI were characterized in order to gain more insight into the molecular evolution of LinB towards β-HCH degradation activity. Since the LinBUT I134V/A247H (=M2-1) mutant showed only weak LinBMI-type activity in our previous study (Ito et al. [2007]), cumulative mutations at the positions A112, I138, and M253 were introduced into the M2-1 mutant. On the basis of kinetic analyses of the resulting three-, four-, and five-point LinBUT mutants (Figures 3, 4 and Table 1), enhancement of the catalytic performance of LinBUT towards β-HCH is discussed.

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