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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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Defining the interfacesof BFDC monomers. (A) BFDC tetramer. MonomerA is colored green, monomer B orange, monomer C cyan, and monomerD magenta. (B) Cartoon with lines indicating interactions betweenthe monomers (circles) and ThDP (diamonds). The active sites (oneper monomer) are found at the A–B interface. (C–E) A–B,A–C, and A–D interfaces, respectively. Figures basedon data from PDB entry 1BFD.
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fig2: Defining the interfacesof BFDC monomers. (A) BFDC tetramer. MonomerA is colored green, monomer B orange, monomer C cyan, and monomerD magenta. (B) Cartoon with lines indicating interactions betweenthe monomers (circles) and ThDP (diamonds). The active sites (oneper monomer) are found at the A–B interface. (C–E) A–B,A–C, and A–D interfaces, respectively. Figures basedon data from PDB entry 1BFD.

Mentions: It is clear from theirrespective X-ray structuresthat KdcA22 is a homodimer and that BFDC18 is a homotetramer (Figure 2). Although ScPDC was initially reported to be ahomotetramer,40 structural analysis suggeststhat its tetrameric form may arise simply by crystal packing.40 To examine this possibility, we turned to theSolvent accessibility-based Protein-Protein Interface iDEntificationand Recognition (SPPIDER) server that can be used to predict the totalsurface area of interfaces from a PDB file.41 Analyses were conducted on three ThDP-dependent decarboxylases,KdcA (PDB entry 2VBF), ScPDC (PDB entry 1PYD), and BFDC (PDB entry 1BFD). In each case,the interface forming the active site, defined as the A–B interface(Figure 2), was clearly identified (Table 1). There was no evidence of any dimer–dimerinterface in the KdcA structure, supporting the crystallographic findingof a homodimer. Interestingly, no evidence was found for any dimer–dimerinterface when the ScPDC structure was used as thesearch model. This reinforces the suggestion that the tetramer observedin the crystal lattice of ScPDC might be an artifactof protein crowding at higher enzyme concentrations. Conversely, analysisof the X-ray structure of BFDC with SPPIDER indicates a robust dimer–dimerinterface accounting for almost 10% of the total surface are of theenzyme (Table 1).


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

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

Defining the interfacesof BFDC monomers. (A) BFDC tetramer. MonomerA is colored green, monomer B orange, monomer C cyan, and monomerD magenta. (B) Cartoon with lines indicating interactions betweenthe monomers (circles) and ThDP (diamonds). The active sites (oneper monomer) are found at the A–B interface. (C–E) A–B,A–C, and A–D interfaces, respectively. Figures basedon data from PDB entry 1BFD.
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fig2: Defining the interfacesof BFDC monomers. (A) BFDC tetramer. MonomerA is colored green, monomer B orange, monomer C cyan, and monomerD magenta. (B) Cartoon with lines indicating interactions betweenthe monomers (circles) and ThDP (diamonds). The active sites (oneper monomer) are found at the A–B interface. (C–E) A–B,A–C, and A–D interfaces, respectively. Figures basedon data from PDB entry 1BFD.
Mentions: It is clear from theirrespective X-ray structuresthat KdcA22 is a homodimer and that BFDC18 is a homotetramer (Figure 2). Although ScPDC was initially reported to be ahomotetramer,40 structural analysis suggeststhat its tetrameric form may arise simply by crystal packing.40 To examine this possibility, we turned to theSolvent accessibility-based Protein-Protein Interface iDEntificationand Recognition (SPPIDER) server that can be used to predict the totalsurface area of interfaces from a PDB file.41 Analyses were conducted on three ThDP-dependent decarboxylases,KdcA (PDB entry 2VBF), ScPDC (PDB entry 1PYD), and BFDC (PDB entry 1BFD). In each case,the interface forming the active site, defined as the A–B interface(Figure 2), was clearly identified (Table 1). There was no evidence of any dimer–dimerinterface in the KdcA structure, supporting the crystallographic findingof a homodimer. Interestingly, no evidence was found for any dimer–dimerinterface when the ScPDC structure was used as thesearch model. This reinforces the suggestion that the tetramer observedin the crystal lattice of ScPDC might be an artifactof protein crowding at higher enzyme concentrations. Conversely, analysisof the X-ray structure of BFDC with SPPIDER indicates a robust dimer–dimerinterface accounting for almost 10% of the total surface are of theenzyme (Table 1).

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