(3] Purification and characterization of leader peptidase from Escherichia coli

doi:10.1016/0076-6879(83)97116-1

Methods in Enzymology

Volume 97, 1983, Pages 40-46

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This chapter discusses the purification protocol and the properties of the leader peptidase from Escherichia coli. The activity of E. coli leader peptidase is determined by measuring the conversion of procoat, the precursor to the major coat protein of bacteriophage M13, to its mature form. Radiolabeled procoat is synthesized in an in vitro protein synthetic reaction in the presence of [³⁵S]methionine using messenger RNA (mRNA) from M13-infected cells to direct the synthesis. The purification of leader peptidase to homogeneity and its characterization have led to a deeper understanding of the events occurring during membrane assembly. The correct processing of precursor proteins by the purified enzyme demonstrates that no cofactors, accessory proteins, or special environmental conditions are necessary for processing to occur. Peptidase is found in both the inner and outer membranes. It is the only known protein found in both membranes that may reflect its role in membrane assembly.

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Type I signal peptidases of Gram-positive bacteria
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Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.
Co-expression of the Bordetella pertussis leader peptidase I results in enhanced processing and expression of the pertussis toxin S1 subunit in Escherichia coli
2000, FEMS Microbiology Letters
Bordetella pertussis is the causative agent of whooping cough. Traditional vaccines against this disease are inherently reactogenic, thus research is currently focussed on the production of less reactive, acellular vaccines. Expression of candidate antigens for these vaccines in Escherichia coli would be preferable, however, several B. pertussis antigens undergo incorrect post-translational processing in E. coli. The leader peptidase gene (lep) of B. pertussis encodes a protein of 294 amino acid residues that shares homology with other prokaryote leader peptidase I sequences. Hydrophilicity analysis based on the predicted amino acid sequence has demonstrated a similar membrane topology to that of E. coli and Salmonella typhimurium leader peptidase I. Co-expression of the B. pertussis lep gene in E. coli strain TOPP2 expressing the pertussis toxin S1 subunit was found to markedly increase the expression and post-translational processing of the S1 protein.
The structure and mechanism of bacterial type I signal peptidases: A novel antibiotic target
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Type I signal peptidases are essential membrane-bound serine proteases that function to cleave the amino-terminal signal peptide extension from proteins that are translocated across biological membranes. The bacterial signal peptidases are unique serine proteases that utilize a Ser/Lys catalytic dyad mechanism in place of the classical Ser/His/Asp catalytic triad mechanism. They represent a potential novel antibiotic target at the bacterial membrane surface. This review will discuss the bacterial signal peptidases that have been characterized to date, as well as putative signal peptidase sequences that have been recognized via bacterial genome sequencing. We review the investigations into the mechanism of Escherichia coli and Bacillus subtilis signal peptidase, and discuss the results in light of the recent crystal structure of the E. coli signal peptidase in complex with a β-lactam-type inhibitor. The proposed conserved structural features of Type I signal peptidases give additional insight into the mechanism of this unique enzyme.
Development of an internally quenched fluorescent substrate for Escherichia coli leader peptidase
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Escherichia colileader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine–lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO₂)–F–S–A–S–A–L–A–K–I–K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively byE. colileader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A–K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 μM) and thek_cat/K_mwas calculated to be 71.1 M⁻¹s⁻¹. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer forE. colileader peptidase.

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(3] Purification and characterization of leader peptidase from Escherichia coli

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J. Biol. Chem.

Cell

Virology

Nature (London)