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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

Structure of LinBMI (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 LinBMI and LinBUT: V134 and V112 (in magenta), L138, H247, and I253 (in cyan), T135 (in green), and T81 (in blue). See text for detail.
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Figure 2: Structure of LinBMI (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 LinBMI and LinBUT: V134 and V112 (in magenta), L138, H247, and I253 (in cyan), T135 (in green), and T81 (in blue). See text for detail.

Mentions: HLDs belong to the α/β-hydrolase family, and their catalytic mechanism consists of the following steps: substrate binding, cleavage of the carbon-halogen bond in the substrate and simultaneous formation of an intermediate covalently bound to a nucleophile, hydrolysis of the alkyl-enzyme intermediate, and release of halide ion and alcohol (Damborsky and Koca [1999]; Janssen [2004]; Prokop et al. [2003]). LinBMI isolated from Sphingobium sp. MI1205 (Ito et al. [2007]) and LinBUT each consist of 296 amino acid residues and share 98% sequence identity, with only seven different amino acid residues between them, at the positions 81, 112, 134, 135, 138, 247, and 253 (Figure 2). However, these two enzymes exhibit significantly different enzymatic behaviors in β-HCH degradation (Figure 1). LinBMI catalyzes the two-step dehalogenation and converts β-HCH to 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and then to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) (LinBMI-type activity) (Ito et al. [2007]), whereas LinBUT catalyzes only the former step (Nagata et al. [2005]) (Figure 1). Furthermore, LinBMI can catalyze the first conversion step an order of magnitude more rapidly than LinBUT (Ito et al. [2007]). Substitution of the LinBUT I134 and A247 residues, which form the catalytic pocket, to the LinBMI-type V and H residues, respectively, resulted in only a weak effect on LinBMI-type activity (Ito et al. [2007]). Additionally, the reciprocal double mutant of LinBMI (V134I/H247A) still retained relatively high LinBMI-type activity (Ito et al. [2007]). These results indicated that one or more of the five other residues are also important for LinBMI-type activity. Our previous site-directed mutagenesis and X-ray crystallographic studies of LinBMI (Okai et al. [2013]) indicated that (i) these five residues are not essential to the LinBMI-type activity, but they all significantly contribute to this activity, and (ii) three of the five residues, V112, L138, and I253, are more important than T81 and T135 for the conversion of PCHL. The structural basis for the importance of the seven amino acid residues of LinBMI can be partially explained by analysis of its tertiary structure (Figure 2). V134 and V112 are located at the catalytic pocket near the nucleophile residue (D108) and at the bottom of the substrate binding pocket, respectively, while L138, H247, and I253 are located at the access tunnels to the catalytic pocket. Therefore, these five amino acid residues may be directly involved in LinBMI-type activity (Okai et al. [2013]). The effect of T135 on LinBMI-type activity may be due to its interaction with I253. However, it is unclear how T81, which is located at the protein surface and far from the active site, affect the activity.


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)

Structure of LinBMI (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 LinBMI and LinBUT: V134 and V112 (in magenta), L138, H247, and I253 (in cyan), T135 (in green), and T81 (in blue). See text for detail.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Structure of LinBMI (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 LinBMI and LinBUT: V134 and V112 (in magenta), L138, H247, and I253 (in cyan), T135 (in green), and T81 (in blue). See text for detail.
Mentions: HLDs belong to the α/β-hydrolase family, and their catalytic mechanism consists of the following steps: substrate binding, cleavage of the carbon-halogen bond in the substrate and simultaneous formation of an intermediate covalently bound to a nucleophile, hydrolysis of the alkyl-enzyme intermediate, and release of halide ion and alcohol (Damborsky and Koca [1999]; Janssen [2004]; Prokop et al. [2003]). LinBMI isolated from Sphingobium sp. MI1205 (Ito et al. [2007]) and LinBUT each consist of 296 amino acid residues and share 98% sequence identity, with only seven different amino acid residues between them, at the positions 81, 112, 134, 135, 138, 247, and 253 (Figure 2). However, these two enzymes exhibit significantly different enzymatic behaviors in β-HCH degradation (Figure 1). LinBMI catalyzes the two-step dehalogenation and converts β-HCH to 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and then to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) (LinBMI-type activity) (Ito et al. [2007]), whereas LinBUT catalyzes only the former step (Nagata et al. [2005]) (Figure 1). Furthermore, LinBMI can catalyze the first conversion step an order of magnitude more rapidly than LinBUT (Ito et al. [2007]). Substitution of the LinBUT I134 and A247 residues, which form the catalytic pocket, to the LinBMI-type V and H residues, respectively, resulted in only a weak effect on LinBMI-type activity (Ito et al. [2007]). Additionally, the reciprocal double mutant of LinBMI (V134I/H247A) still retained relatively high LinBMI-type activity (Ito et al. [2007]). These results indicated that one or more of the five other residues are also important for LinBMI-type activity. Our previous site-directed mutagenesis and X-ray crystallographic studies of LinBMI (Okai et al. [2013]) indicated that (i) these five residues are not essential to the LinBMI-type activity, but they all significantly contribute to this activity, and (ii) three of the five residues, V112, L138, and I253, are more important than T81 and T135 for the conversion of PCHL. The structural basis for the importance of the seven amino acid residues of LinBMI can be partially explained by analysis of its tertiary structure (Figure 2). V134 and V112 are located at the catalytic pocket near the nucleophile residue (D108) and at the bottom of the substrate binding pocket, respectively, while L138, H247, and I253 are located at the access tunnels to the catalytic pocket. Therefore, these five amino acid residues may be directly involved in LinBMI-type activity (Okai et al. [2013]). The effect of T135 on LinBMI-type activity may be due to its interaction with I253. However, it is unclear how T81, which is located at the protein surface and far from the active site, affect the activity.

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