title: 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 phenyl alanine 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 k(cat)/K(m) 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.
authors.affiliation: College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Yep
authors.forename: George L
authors.initials: GL
authors.lastname: Kenyon
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
mesh_terms: D000596:Amino Acids; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase