Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

Abstract

Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenylalanine with only a 5-fold decrease in k cat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues. catalytic mechanism ͉ thiamin diphosphate

Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…More recently, McLeish and coworkers have been carrying out saturation mutagenesis experiments [17] probing the function of two active center histidine residues (His70 and His281) on benzoylformate decarboxylase (BFDC), long believed to participate in acid-base reactions [18]. Surprisingly, their results indicated that hydrophobic residues could replace the His281 with little penalty, and even with His70 there was only a 70-fold penalty on k cat /K m .…”

The kinetic characterization and X-ray structure of a putative benzoylformate decarboxylase from M. smegmatis highlights the difficulties in the functional annotation of ThDP-dependent enzymes

“…This was in keeping with the proposed role of His281 in catalysis [10]. However, in a later saturation mutagenesis study, it was found that several residues incapable of acting as a general acid could replace His281 with only a modest decrease in activity [13]. In particular, the H281Y variant displayed only an 84-fold decrease in k cat /K m and this was largely due to a 46-fold decrease in k cat value [13].…”

“…Successful mutagenesis was indicated by the presence of new XmaI and XmnI restriction sites for the Y281H and I466A variants, respectively. The mutations, and the absence of extraneous The PpBFDC H281Y and A460I variants were available from previous studies [12,13]. The H281Y/A460I double mutant was prepared by PCR mutagenesis on pET24PpBFDC_H281Y-His [13] followed by screening for loss of a BanI restriction site.…”

“…1A. Through extensive mutagenic, kinetic and crystallographic studies, Ser26, Glu47, His70 and His281 have been identified as the major players in assisting the ThDP cofactor in the decarboxylation of benzoylformate [10,11,13,25,26]. In the context of the reaction mechanism shown in Scheme 1, Glu47 helps to stabilize the 1,4-imino tautomer of ThDP that is formed prior to ylide formation.…”

“…One significant exception is the benzoylformate decarboxylase (BFDC) isolated from Pseudomonas putida (PpBFDC), which has the innate ability to synthesize mixed acetoin-like products in the S-configuration. There has been considerable interest in (i) understanding which PpBFDC residues are absolutely required for catalytic activity, (ii) identifying those residues controlling the substrate specify for both the donor and acceptor aldehydes, and (iii) identifying other BFDCs that will provide S-products [7][8][9][10][11][12][13][14]. It is anticipated that this knowledge will lead to the development of biocatalysts capable of generating novel products.…”