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Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

Andrews FH, Rogers MP, Paul LN, McLeish MJ - Biochemistry (2014)

Bottom Line: Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity.It was also found to be catalytically inactive.Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana 46202, United States.

ABSTRACT
The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

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Equilibrium unfolding of BFDC by urea.(A) Relative changes inintrinsic fluorescence at 327 nm (λex at 280 nm)for wt BFDC (●) and the R141E/A306F variant(◇). (B) Relative changes in ellipticity at 222 nm (●)and enzymatic activity (○) for wt BFDC. Allchanges were monitored at 20 °C. The results shown are the averageof at least two independent measurements.
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fig6: Equilibrium unfolding of BFDC by urea.(A) Relative changes inintrinsic fluorescence at 327 nm (λex at 280 nm)for wt BFDC (●) and the R141E/A306F variant(◇). (B) Relative changes in ellipticity at 222 nm (●)and enzymatic activity (○) for wt BFDC. Allchanges were monitored at 20 °C. The results shown are the averageof at least two independent measurements.

Mentions: The urea-mediatedunfolding of wt BFDC and the R141E/A306F variantwas followed by both intrinsic fluorescence and CD spectroscopy. Tohighlight any differences, the unfolding experiments were conductedat an enzyme concentration of 0.15 mg/mL. At this concentration, the wt enzyme is tetrameric whereas the R141E/A306F variantis expected to be primarily dimeric. As shown in Figure 6A, when monitored using the intrinsic fluorescence signalat 327 nm, the unfolding of wt BFDC appears to havethree transitions. Initially, between 0 and 0.5 M urea, there is asmall increase in fluorescence. Another small transition is observedon going from 0.5 to 2 M urea, while the largest transition takesplace between 2 and 6 M urea. Potentially, the first corresponds tothe dissociation of tetramers to dimers; the second is that of thedimers to monomers, and the third results from the unfolding of themonomers. Such a pattern was also observed for ScPDC.25 By contrast, the unfolding of theR141E/A306F variant showed only two transitions (Figure 6A), most likely due to the initial unfolding of the dimerfollowed by unfolding of the monomer.


Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

Andrews FH, Rogers MP, Paul LN, McLeish MJ - Biochemistry (2014)

Equilibrium unfolding of BFDC by urea.(A) Relative changes inintrinsic fluorescence at 327 nm (λex at 280 nm)for wt BFDC (●) and the R141E/A306F variant(◇). (B) Relative changes in ellipticity at 222 nm (●)and enzymatic activity (○) for wt BFDC. Allchanges were monitored at 20 °C. The results shown are the averageof at least two independent measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215898&req=5

fig6: Equilibrium unfolding of BFDC by urea.(A) Relative changes inintrinsic fluorescence at 327 nm (λex at 280 nm)for wt BFDC (●) and the R141E/A306F variant(◇). (B) Relative changes in ellipticity at 222 nm (●)and enzymatic activity (○) for wt BFDC. Allchanges were monitored at 20 °C. The results shown are the averageof at least two independent measurements.
Mentions: The urea-mediatedunfolding of wt BFDC and the R141E/A306F variantwas followed by both intrinsic fluorescence and CD spectroscopy. Tohighlight any differences, the unfolding experiments were conductedat an enzyme concentration of 0.15 mg/mL. At this concentration, the wt enzyme is tetrameric whereas the R141E/A306F variantis expected to be primarily dimeric. As shown in Figure 6A, when monitored using the intrinsic fluorescence signalat 327 nm, the unfolding of wt BFDC appears to havethree transitions. Initially, between 0 and 0.5 M urea, there is asmall increase in fluorescence. Another small transition is observedon going from 0.5 to 2 M urea, while the largest transition takesplace between 2 and 6 M urea. Potentially, the first corresponds tothe dissociation of tetramers to dimers; the second is that of thedimers to monomers, and the third results from the unfolding of themonomers. Such a pattern was also observed for ScPDC.25 By contrast, the unfolding of theR141E/A306F variant showed only two transitions (Figure 6A), most likely due to the initial unfolding of the dimerfollowed by unfolding of the monomer.

Bottom Line: Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity.It was also found to be catalytically inactive.Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana 46202, United States.

ABSTRACT
The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

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