Poster abstracts

Poster number 70 submitted by Peng Wang

On the catalytic mechanism of the E.coli signal peptide peptidase A

Peng Wang (Chemistry), Eunjung Shim (Chemistry)

Abstract:
The Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein, but does not label the mutant S409A with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine K209 in the N-terminal-domain that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor, and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that the E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.

Keywords: SppA, mechanism, topology