Browsing by Subject "Benzoylformate decarboxylase (BFDC)"

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    Rates and Intermediates in Ser26 Mutants of Benzoylformate Decarboxylase
    (Office of the Vice Chancellor for Research, 2016-04-08) Lian, NiLen T.; McLeish, Michael J.; Ray, Bruce D.
    Benzoylformate decarboxylase (BFDC), a thiamine diphosphate dependent enzyme, catalyzes decarboxylation of benzoylformate to benzaldehyde and CO2. The BFDC reaction proceeds through at least four individual chemical steps and, recently, NMR spectroscopy has been used to measure the ratios of intermediates in the overall reaction. This method permits calculation of rate constants for formation of the first intermediate, mandelylThDP (k2) and its subsequent decarboxylation (k3), as well as the combined breakdown of the enamine and product release (k4). As part of a study of the contributions of the active site residues, Ser26, His70 and His281, to the individual catalytic steps several Ser26 variants were expressed and purified. Initially, the variants were characterized using steady-state kinetics. Subsequently, the enzymes were mixed with benzoylformate and the mixture immediately acid quenched to trap intermediates of the reaction. NMR spectroscopy was used to identify and quantitate individual catalytic intermediates. Rate constants for the formation of these intermediates were then determined and compared to those of the wild-type enzyme. Here we report those results and discuss their implications for the role of Ser26 in the BFDC reaction mechanism.
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    Using saturation mutagenesis to explore substrate specificity and catalysis in benzoylformate decarboxylase
    (Office of the Vice Chancellor for Research, 2010-04-09) McLeish, Michael J.; Andrews, Forest; Horton, Joshua; Yep, Alejandra
    Benzoylformate decarboxylase (BFDC) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Zymomonas mobilis are thiamin diphosphate (ThDP)-dependent enzymes. The two share a common three-dimensional structure and catalyze a similar chemical reaction, i.e., decarboxylation of 2-keto acids. However, they vary significantly in their substrate utilization pattern. In particular, BFDC has extremely limited activity with pyruvate, while PDC has no activity with benzoylformate. Both enzymes also catalyze stereospecific carboligation reactions that are of commercial interest, again with a different range of substrates. In order to identify similarities and differences on a molecular level, and to reveal factors responsible for substrate specificity and enantioselectivity, the X-ray structures BFDC and PDC were compared. Residues identified in this process were subjected to site-directed mutagenesis. The results show that, although it was not possible to simply interchange substrates, it was possible to engineer enzymes that had distinctly different substrate specificities while retaining excellent kinetic activity. However, it also became apparent that a more general approach was needed. Towards this end we developed a screening procedure for BFDC to enable us to use saturation mutagenesis to examine residues involved in substrate specificity. During the development of the methodology it became clear that it was possible to use this approach to explore residues involved in catalysis by BFDC. Here we describe the unexpected results obtained using saturation mutagenesis on putative catalytic residues. In addition we report towards converting BFDC into an efficient pyruvate decarboxylase.