Signal peptidase (SPase) has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at -3 (P3) position. Structures of SPase reveal candidate residues making up the S1 and S3 pockets that bind the P1 and P3 residues of the preprotein substrate. We have used various methods to examine the importance of these residues in promoting cleavage of the pro-OmpA-nuclease A (PONA) substrate with WT and mutant processing regions. We find that the S1 and S3 binding sites can tolerate changes without great effect on activity. Changing the I144 residue to cysteine results in cleavage at multiple sites. Also, we find that SPase is able to cleave after -1F in an I144A/I86A double mutant. Additionally, alteration of I144 and I86 to the corresponding residues in the homologous Imp1 protease changes the specificity to promote cleavage following –1N. This work shows that I144 and I86 contribute to SPase substrate specificity and that I144 is important for the accuracy of the cleavage reaction. Studies with corresponding β-lactamase substrate mutants were used to investigate the altered specificity revealed in the PONA studies. Using β-lactamase as a substrate allows for an antibiotic selection method to assay processing in vivo. A two plasmid system was developed to assay combinations of SPase and β-lactamase substrate mutants. This study assayed growth of the TS SPase strain, IT41, containing various combinations of SPase and β-lactamase mutants. Growth under conditions of high ampicillin suggests efficient processing of β-lactamase. Growth was observed in some cases when the β-lactamase substrate carried a relatively conservative -1V substitution in place of the WT -1A residue. The two-plasmid system utilizing the β-lactamase substrate developed in these studies has potential to be implemented in an unbiased genetic selection technique to further define the substrate specificity determinants of signal peptidase. β-lactamase processing by SPase mutants that affected specificity in the PONA studies was also assayed by pulse-chase. These studies confirm the results observed with the PONA substrate. One added benefit of using β-lactamase as a substrate is that the signal peptide cleavage region possesses no alternate cleavage sites.
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/09(E), Dissertation Abstracts International|
|Keywords:||Beta-lactamase, I144a, I144c/i86c, Mutant, Pona, Spase|
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