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Using saturation mutagenesis to replace putative catalytic residues in thiamin diphosphate dependent enzymes
Author(s) -
Yep Alejandra,
Kenyon George L,
McLeish Michael J
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1016-b
Subject(s) - saturated mutagenesis , chemistry , enzyme kinetics , biochemistry , enzyme , decarboxylation , mutagenesis , stereochemistry , site directed mutagenesis , protonation , pseudomonas putida , residue (chemistry) , enzyme catalysis , catalysis , active site , organic chemistry , mutant , ion , gene
Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamin diphosphate (ThDP)‐dependent enzyme that carries out the non‐oxidative decarboxylation of aromatic 2‐keto carboxylic acids. Initially we developed a screening procedure to enable us to use saturation mutagenesis to examine residues involved in substrate specificity. However, it also proved possible to use this methodology to explore residues involved in catalysis. Earlier studies, using site‐directed mutagenesis have implicated Ser26, His70 and His281 as playing important roles in the catalytic mechanism of BFD. Here we describe the screening procedure and its use in identifying phenylalanine as a residue that could be used to replace His281 with only a 5‐fold decrease in k cat / K M . This is an intriguing result as previous reports had implicated His281 as being necessary for protonation of the enamine intermediate, a scenario which now needs re‐evaluation. Other interesting results included the observation that Ser26 could be replaced by either threonine or leucine without significant loss of activity. We are currently extending the use of this technique to other ThDP‐dependent enzymes. This work was supported by NSF EF‐0425719.