Benzoylformate decarboxylases (BFDCs) are a relatively uncommon group of thiamin diphosphate-dependent enzymes. As their name suggests, they catalyze the decarboxylation of benzoylformate, with the enzyme from Pseudomonas putida (PpBFDC) being the most extensively studied. Based upon sequence homology, the recently established Thiamin Enzyme Engineering Database (TEED) has identified dozens of sequences in a variety of other microorganisms and annotated them as BFDCs. Interestingly, while the majority of these putative BFDCs share >40% sequence identity with PpBFDC, many lack the amino acids thought to be important in its function. Further, most of the annotated sequences are from organisms with no known pathway involving benzoylformate. To determine the integrity of these sequence annotations, these previously unstudied enzymes must be functionally characterized to determine if they are, in fact, true BFDCs. Currently, we are studying putative BFDCs from Polynucleobacter necessarius and Mycobacterium smegmatis, both of which share most of the catalytic residues as PpBFDC but have alterations in residues involved in substrate specificity. We have successfully expressed and purified these supposed BFDCs, and characterized them by assaying with a variety of both metabolic and unnatural 2-ketoacid substrates. We have also characterized PnBFDC by X-ray crystallography. Comparison of their activity with that of PpBFDC indicates that these two sequences were both incorrectly annotated. We are currently in the process of trying to identify their natural substrates.

Benzoylformate decarboxylase and the annotation problem

246th National Meeting (2013)

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