Abstract
BackgroundThe 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 β turn at the base of the active site zinc binding region.ResultsTo 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.ConclusionAlthough 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.
Highlights
The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases
Due to the difficulty of isolating and handling sufficient quantities of this truncated version of AprA, attention was focused on overproduction of the full-length mature form of AprA which corresponds to residues G10 to V479
Mature full-length AprA and DFM incorporation The gene for the full-length mature form of AprA [24,25,26] which corresponds to residues G10 to V479 was isolated on to pET22b and overexpressed in E. coli BL21, resulting in inclusion body formation
Summary
The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. As diverse as the members of this superfamily are, surprisingly they all share a structurally similar catalytic site which includes a common zinc binding motif and an absolutely conserved methionine-containing 1,4-tight β turn, termed the "Met-turn" in their active sites [4]. The matrixins, or matrix metalloproteinases (MMPs) include the collagenases, gelatinases and stromelysins [5,6]. Members of this subfamily degrade extracellular matrix for tissue remodeling and maturation, but they have been found to release and activate several growth factors and modulate chemotactic signals [5]. They have been implicated in promoting tumour invasion and metastasis by degradation of matrix barriers, exacerbating periodontal disease,
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