Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease.
BACKGROUND
The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight beta turn at the base of the active site zinc binding region.


RESULTS
To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme.


CONCLUSIONS
Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.
Walasek
Paula
P
Department of Chemistry, University of Waterloo, 200 University Avenue, Waterloo, Ontario, Canada N2L 3G1. pwalasek@scimail.uwaterloo.ca
Honek
John F
JF
D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics*; D008715:Methionine / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D008715:Methionine; D010450:Endopeptidases; D008666:Metalloendopeptidases; C087822:alkaline protease