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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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Leu109and Leu110 are positioned by the interaction of Arg141 andArg120 of monomer A with Glu107 and Asp114 of monomer C, respectively.Blue dashes indicate distances of ∼3 Å. Red dashes indicatedistances of ≤5 Å.
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fig9: Leu109and Leu110 are positioned by the interaction of Arg141 andArg120 of monomer A with Glu107 and Asp114 of monomer C, respectively.Blue dashes indicate distances of ∼3 Å. Red dashes indicatedistances of ≤5 Å.

Mentions: Examination of the X-ray structure of BFDC in complex withbenzoylphosphonic acid methyl ester (MBP), an analogue of benzoylformate,shows that Leu109 lines the phenyl-binding pocket of BFDC and thatLeu110 has five points of contact within 5 Å of the glyoxylateanalogue portion of MBP (Figure 9). From thisstructure, it appears that Leu110 acts as a clamp to lock the glyoxylatemoiety into the perpendicular arrangement of the carboxylate groupto the thiazolium–C2α bond. This geometry is thoughtto promote decarboxylation by allowing the maximal overlap of theπ electrons of the thiazolium ring and the p orbital of thescissile bond.52,53 Intriguingly, the active siteloop of BFDC is bookended by Glu107 and Asp114, which, of course,interact with Arg141 and Arg120, respectively, in the A–C interface(Figure 4). It is not unreasonable to imaginethat these two salt bridges may be responsible for maintaining thecorrect positioning of Leu109 and Leu110 within the active site ofBFDC, thereby contributing to both the overall hydrophobicity of theactive site and the correct position of the substrate within it.


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

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

Leu109and Leu110 are positioned by the interaction of Arg141 andArg120 of monomer A with Glu107 and Asp114 of monomer C, respectively.Blue dashes indicate distances of ∼3 Å. Red dashes indicatedistances of ≤5 Å.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215898&req=5

fig9: Leu109and Leu110 are positioned by the interaction of Arg141 andArg120 of monomer A with Glu107 and Asp114 of monomer C, respectively.Blue dashes indicate distances of ∼3 Å. Red dashes indicatedistances of ≤5 Å.
Mentions: Examination of the X-ray structure of BFDC in complex withbenzoylphosphonic acid methyl ester (MBP), an analogue of benzoylformate,shows that Leu109 lines the phenyl-binding pocket of BFDC and thatLeu110 has five points of contact within 5 Å of the glyoxylateanalogue portion of MBP (Figure 9). From thisstructure, it appears that Leu110 acts as a clamp to lock the glyoxylatemoiety into the perpendicular arrangement of the carboxylate groupto the thiazolium–C2α bond. This geometry is thoughtto promote decarboxylation by allowing the maximal overlap of theπ electrons of the thiazolium ring and the p orbital of thescissile bond.52,53 Intriguingly, the active siteloop of BFDC is bookended by Glu107 and Asp114, which, of course,interact with Arg141 and Arg120, respectively, in the A–C interface(Figure 4). It is not unreasonable to imaginethat these two salt bridges may be responsible for maintaining thecorrect positioning of Leu109 and Leu110 within the active site ofBFDC, thereby contributing to both the overall hydrophobicity of theactive site and the correct position of the substrate within it.

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.

Show MeSH