Determination of the Kinetic Parameters of Escherichia coli Leader Peptidase Activity Using a Continuous Assay: The pH Dependence and Time-Dependent Inhibition by .beta.-Lactams Are Consistent with a Novel Serine Protease Mechanism

Kuo, David; Weidner, Jeffrey R.; Griffin, Patrick R.; Shah, Shrenik K.; Knight, Wilson B.

doi:10.1021/bi00193a023

Cited by 49 publications

(33 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5). In addition to their role in blocking transpeptidases responsible for cross-linking peptidoglycan, ␤-lactam antibiotics also inhibit signal peptidase I (22). As a result, ␤-lactams inhibit the proteolytic processing of autotransporters such as PmpD, presumably by preventing their release from the cytoplasmic membrane and transport to the outer membrane (20).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, it was reported that the translocation of PmpD to the bacterial cell surface is inhibited by ␤-lactam antibiotics (20), presumably as a result of inhibition of signal peptidase I activity at the cytoplasmic membrane (22). In Chlamydia-infected cells, low-level ␤-lactam antibiotic treatment prevents bacterial division, leading to the formation of enlarged RBs that morphologically resemble persistent forms (26).…”

Section: Identification Of Pls Proteins Ct049 and Ct050mentioning

confidence: 99%

See 1 more Smart Citation

Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

Jørgensen

Valdivia

2008

Infect Immun

View full text Add to dashboard Cite

The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of ␤-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Identification Of Pls Proteins Ct049 and Ct050mentioning

confidence: 99%

Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

Jørgensen

Valdivia

2008

Infect Immun

View full text Add to dashboard Cite

show abstract

“…The latter pre-proteins are not cleaved in E. coli and act as competitive inhibitors of signal peptidase. Recently, after a long search, two pharmaceutical companies have discovered that certain p-lactam compounds inhibit the E. coli signal peptidase (Kuo et al, 1994;Allsop et al, 1995). The best inhibitor reported is a 5s penem derivative, which has an I.C.…”

Section: Substrates and Inhibitorsmentioning

confidence: 99%

The chemistry and enzymology of the type I signal peptidases

et al. 1997

View full text Add to dashboard Cite

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membranebound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.

show abstract

“…The first effective synthetic signal peptidase inhibitors to be discovered were described by Kuo and collegues in 1994 (25). They showed that ␤-lactam analogs inhibited E. coli SPase.…”

mentioning

confidence: 99%

Crystallographic and Biophysical Analysis of a Bacterial Signal Peptidase in Complex with a Lipopeptide-based Inhibitor

Paetzel

Goodall

Kania

et al. 2004

Journal of Biological Chemistry

149

View full text Add to dashboard Cite

We report here the crystallographic and biophysical analysis of a soluble, catalytically active fragment of the Escherichia coli type I signal peptidase (SPase ⌬2-75) in complex with arylomycin A 2 . The 2.5-Å resolution structure revealed that the inhibitor is positioned with its COOH-terminal carboxylate oxygen (O45) within hydrogen bonding distance of all the functional groups in the catalytic center of the enzyme (Ser 90 O-␥, Lys 145 N-, and Ser 88 O-␥) and that it makes ␤-sheet type interactions with the ␤-strands that line each side of the binding site. Ligand binding studies, calorimetry, fluorescence spectroscopy, and stopped-flow kinetics were also used to analyze the binding mode of this unique non-covalently bound inhibitor. The crystal structure was solved in the space group P4 3 2 1 2. A detailed comparison is made to the previously published acyl-enzyme inhibitor complex structure (space group: P2 1 2 1 2) and the apo-enzyme structure (space group: P4 1 2 1 2). Together this work provides insights into the binding of pre-protein substrates to signal peptidase and will prove helpful in the development of novel antibiotics.Type I signal (leader) peptidase (SPase, 1 EC 3.4.21.89) is the membrane-bound serine endopeptidase that catalyzes the cleavage of the amino-terminal signal (or leader) peptide from secretory proteins and some membrane proteins (for recent reviews, see Refs. 1-3). Evolutionarily, SPase belongs to the protease clan SF and the protease family S26 (4). The Escherichia coli SPase has served as the model Gram-negative SPase and is the most thoroughly characterized SPase to date. It has been cloned (5), sequenced (6), overexpressed (7), purified (6,8,9), and kinetically (10), and structurally (11, 12) characterized. E. coli SPase (323 amino acids, 35,988 Da, pI 6.9) contains two amino-terminal transmembrane segments (residues 4 -28 and 58 -76), a small cytoplasmic region (residues 29 -58), and a carboxyl-terminal periplasmic catalytic region (residues 77-323). A catalytically active fragment of SPase (SPase ⌬2-75) corresponding to the periplasmic region (lacking the two transmembrane segments and the cytoplasmic domain) has been cloned, purified, characterized (13,14), and crystallized (15). Interestingly, the ⌬2-75 construct required detergent or lipid for optimal activity (14) and crystallization (15).The crystal structure of ⌬2-75 has been solved in complex with a ␤-lactam-type inhibitor as well in the apo-form (11, 12). The structures of E. coli SPase ⌬2-75 revealed that the periplasmic region of bacterial signal peptidase has a unique, mostly ␤-structure protein fold made of several coiled ␤-sheets and contains an Src homology 3-like barrel. The periplasmic region of SPase is made up of two domains. Domain I contains the catalytic residues and all of the conserved regions of sequence. It also contains an unusually large exposed hydrophobic surface that is consistent with a membrane association surface and possibly the detergent/lipid requirement of the ⌬2-75 deletion construct. The ...

show abstract

Determination of the Kinetic Parameters of Escherichia coli Leader Peptidase Activity Using a Continuous Assay: The pH Dependence and Time-Dependent Inhibition by .beta.-Lactams Are Consistent with a Novel Serine Protease Mechanism

Cited by 49 publications

References 64 publications

Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

The chemistry and enzymology of the type I signal peptidases

Crystallographic and Biophysical Analysis of a Bacterial Signal Peptidase in Complex with a Lipopeptide-based Inhibitor

Contact Info

Product

Resources

About