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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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Interactionsat the A–C interface. View of charged residueshighlighting those with interatomic distances of ∼3 Å.
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fig4: Interactionsat the A–C interface. View of charged residueshighlighting those with interatomic distances of ∼3 Å.

Mentions: At ∼500 Å2, the A–C interface accountsfor ∼2.5% of the total surface area of monomer A and is mostlycomprised of residues in charge–charge interactions. The sidechains of Glu107 and Asp114 of monomer A, for example, form salt bridgeswith Arg141 and Arg120, respectively, of monomer C (Figure 4). Because of the 2-fold symmetry, Arg120 and Arg141of monomer A also interact with Asp114 and Glu107 of monomer C. Thesefour positions were selected as targets for mutagenesis, specificallyto introduce like-charge repulsions in an attempt to destabilize thedimer–dimer interface. A summary of the various interactions,and the rationale for the mutagenesis experiments can be found inTable S2 of the Supporting Information.


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

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

Interactionsat the A–C interface. View of charged residueshighlighting those with interatomic distances of ∼3 Å.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Interactionsat the A–C interface. View of charged residueshighlighting those with interatomic distances of ∼3 Å.
Mentions: At ∼500 Å2, the A–C interface accountsfor ∼2.5% of the total surface area of monomer A and is mostlycomprised of residues in charge–charge interactions. The sidechains of Glu107 and Asp114 of monomer A, for example, form salt bridgeswith Arg141 and Arg120, respectively, of monomer C (Figure 4). Because of the 2-fold symmetry, Arg120 and Arg141of monomer A also interact with Asp114 and Glu107 of monomer C. Thesefour positions were selected as targets for mutagenesis, specificallyto introduce like-charge repulsions in an attempt to destabilize thedimer–dimer interface. A summary of the various interactions,and the rationale for the mutagenesis experiments can be found inTable S2 of the Supporting Information.

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