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Kinetic and sequence-structure-function analysis of LinB enzyme variants with β- and δ-hexachlorocyclohexane.

Pandey R, Lucent D, Kumari K, Sharma P, Lal R, Oakeshott JG, Pandey G - PLoS ONE (2014)

Bottom Line: Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention.One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane.Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Ecosystem Sciences, Acton, ACT, Australia.

ABSTRACT
Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

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Comparative conformational flexibility within the cap domain of two LinB variants.Sausage diagram for LinBB90A (A) and LinBG2.2 (B) in which the thickness of the chain as well as the colour denotes the average RMSD of each residue. The active site catalytic residues are rendered in ball and stick. The RMSD of each residue as a function of time is plotted for LinBB90A (C) and LinBG2.2 (D).
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pone-0103632-g001: Comparative conformational flexibility within the cap domain of two LinB variants.Sausage diagram for LinBB90A (A) and LinBG2.2 (B) in which the thickness of the chain as well as the colour denotes the average RMSD of each residue. The active site catalytic residues are rendered in ball and stick. The RMSD of each residue as a function of time is plotted for LinBB90A (C) and LinBG2.2 (D).

Mentions: Molecular dynamics simulations were used to probe the conformational dynamics of the synthetic LinBG2.2 variant relative to LinBB90A (our reference variant). These simulations revealed large differences in conformational flexibility within the cap domain (Figure 1). This domain occludes the active site and has previously been shown to be more flexible in LinB relative to the related dehalogenases DhaA and DhlA [28]. Here we see that LinBG2.2 shows greatly enhanced flexibility in this region when compared to LinBB90A. Unlike some of the other variants of this enzyme, LinBB90A has a histidine at position 247. This residue has been hypothesized to enhance activity by reducing solvent access to the active site [13]. Although this mechanism may be feasible for some of the smaller haloalkane dehalogenase substrates (such as dichloroethane and trichloropropane), HCH cannot easily diffuse into the partially occluded active site in LinB, nor can it easily enter through the side tunnels previously characterized for this enzyme [13]. As such, it seems reasonable to assume that increased flexibility in the cap domain plays an important role in the enhancement of catalysis as observed in LinBG2.2 relative to LinBB90A by removing kinetic barriers to substrate binding (in spite of the fact that this potentially allows greater solvent penetration into the active site).


Kinetic and sequence-structure-function analysis of LinB enzyme variants with β- and δ-hexachlorocyclohexane.

Pandey R, Lucent D, Kumari K, Sharma P, Lal R, Oakeshott JG, Pandey G - PLoS ONE (2014)

Comparative conformational flexibility within the cap domain of two LinB variants.Sausage diagram for LinBB90A (A) and LinBG2.2 (B) in which the thickness of the chain as well as the colour denotes the average RMSD of each residue. The active site catalytic residues are rendered in ball and stick. The RMSD of each residue as a function of time is plotted for LinBB90A (C) and LinBG2.2 (D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116220&req=5

pone-0103632-g001: Comparative conformational flexibility within the cap domain of two LinB variants.Sausage diagram for LinBB90A (A) and LinBG2.2 (B) in which the thickness of the chain as well as the colour denotes the average RMSD of each residue. The active site catalytic residues are rendered in ball and stick. The RMSD of each residue as a function of time is plotted for LinBB90A (C) and LinBG2.2 (D).
Mentions: Molecular dynamics simulations were used to probe the conformational dynamics of the synthetic LinBG2.2 variant relative to LinBB90A (our reference variant). These simulations revealed large differences in conformational flexibility within the cap domain (Figure 1). This domain occludes the active site and has previously been shown to be more flexible in LinB relative to the related dehalogenases DhaA and DhlA [28]. Here we see that LinBG2.2 shows greatly enhanced flexibility in this region when compared to LinBB90A. Unlike some of the other variants of this enzyme, LinBB90A has a histidine at position 247. This residue has been hypothesized to enhance activity by reducing solvent access to the active site [13]. Although this mechanism may be feasible for some of the smaller haloalkane dehalogenase substrates (such as dichloroethane and trichloropropane), HCH cannot easily diffuse into the partially occluded active site in LinB, nor can it easily enter through the side tunnels previously characterized for this enzyme [13]. As such, it seems reasonable to assume that increased flexibility in the cap domain plays an important role in the enhancement of catalysis as observed in LinBG2.2 relative to LinBB90A by removing kinetic barriers to substrate binding (in spite of the fact that this potentially allows greater solvent penetration into the active site).

Bottom Line: Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention.One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane.Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Ecosystem Sciences, Acton, ACT, Australia.

ABSTRACT
Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

Show MeSH