Biochemical characterization of signal peptide processing enzymes: Staphylococcus aureus signal peptidase I and Escherichia coli signal peptide peptidase A2

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Signal peptidase I
Signal peptide peptidase A2
Ser/Lys proteases
Enzyme kinetics
Enzyme/substrate interactions
Protein translocation

Type I signal peptidase, SPase I, is an essential bacterial enzyme participating in the process of protein secretion. SPase I catalyzes the conversion of pre-proteins to mature proteins by cleaving off the amino-terminal signal peptides from the pre-proteins during protein secretion. The removal of these remnant signal peptides, required for the continuation of the secretion process, is not a well understood process in bacteria. In Escherichia coli, signal peptide peptidase A, SppA, together with other enzymes, is responsible for the removal of these remnant signal peptides. This thesis work focuses on characterizing and comparing Staphylococcus aureus SPase I, SpsB, with other bacterial SPase I as well as characterizing a previously unexamined SppA related enzyme, E. coli signal peptide peptidase A2, SppA2. A fluorescent lipidated peptide substrate with a Gram-positive signal peptide sequence was used to characterize the Michaelis-Menten kinetic constants for S. aureus SpsB along with SPase I from Bacillus subtilis, Staphylococcus epidermidis, and E. coli. E. coli SPase I has a significantly lower catalytic efficiency towards the substrate. A previously characterized E. coli pre-protein was mutated to match the sequence of the Gram-positive peptide sequence, leading to a significantly reduced maturation rate by E. coli SPase I, both in vitro and in vivo. These results have led to the discovery of a previously uncharacterized residue in the SPase I substrate binding groove, proline 88 in E. coli, which may contribute to the difference in catalytic efficiency observed between Gram-positive and Gram-negative SPase I enzymes. Limited proteolysis has revealed that E. coli SppA2 has an N-terminal protease sensitive region, residues 39 to 91, and a C-terminal trypsin resistant ,trSppA2, domain, residues 92 to 349. Light scattering results indicate that trSppA2 forms octamers in solution with a proposed dome-shape structure similar to that of E. coli and B. subtilis SppA. Activity and mutagenesis studies demonstrate that trSppA2 can digest both small peptides and folded proteins, with a preference for hydrophobic substrates, while the S178A and K230A mutants are inactive suggesting that SppA2 is a serine / lysine dyad enzyme. Lastly, the protease sensitive region is required for proper protein folding and may regulate substrate traffic into and out of the inner cavity of the SppA2 octamer.

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Mark Paetzel
Thesis type: 
(Thesis) Ph.D.