Article pubs.acs.org/biochemistry
Perturbation of the Monomer−Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer Forest H. Andrews,† Megan P. Rogers,† Lake N. Paul,‡ and Michael J. McLeish*,† †
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202, United States ‡ Bindley Biosciences Center, Discovery Park, Purdue University, West Lafayette, Indiana 47904, United States S Supporting Information *
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
