Inhibition by penem of processing peptidases from cyanobacteria and chloroplast thylakoids

Barbrook, Adrian C.; Packer, Jeremy C.L.; Howe, Christopher J.

doi:10.1016/s0014-5793(96)01239-2

Cited by 17 publications

(10 citation statements)

References 23 publications

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“…An important fact, however, is that the catalytic function of CtpA was not inhibited by penem, which has been shown to be a potent inhibitor of two kinds of serine protease with a Ser/Lys catalytic dyad, i.e. the E. coli leader peptidase (22,33) and the thylakoidal processing peptidases from cyanobacteria and higher plants (34). This inhibitory effect of penem is reported to be due to covalent binding of the ␤-lactam ring to the catalytic Ser residue (22).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, by taking advantage of the quantity of enzyme overexpressed in E. coli, we systematically examined a wide variety of protease inhibitors (including serine, cysteine, and aspartic protease and metalloprotease inhibitors) at different concentrations for the C-terminal cleavage of the synthetic oligopeptide by thrombin-treated CtpA overexpressed in E. coli. This included penem, which is unique in that it specifically inhibits some of the novel serine proteases possessing the Ser/Lys catalytic dyad (33,34). However, as summarized in Table I, despite thorough investigation, the CtpA activity proved to be resistant to all of the protease inhibitors tested, including penem.…”

Section: Cleavage Of C-terminal Oligopeptides By Ctpa Expressed In Ementioning

confidence: 99%

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Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Yamamoto

Inagaki

Satoh

2001

Journal of Biological Chemistry

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CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions. The D1 protein is a membrane-spanning subunit constituting the core part of the photosystem II (PSII) 1 reaction center (1, 2). In the predicted secondary structure, the D1 protein consists of five transmembrane ␣-helices and a C-terminal extension protruding into the luminal space of thylakoids (reviewed in Ref. 2). A curious feature of this protein is the extraordinarily high rate of metabolic turnover in vivo, despite its fundamental importance in the structure and function of PSII (3). In the dynamic process, the D1 protein, which is encoded by the psbA gene in the plastid genome in the case of eukaryotic organisms, is synthesized on membrane-associated ribosomes on the cytosolic or stromal surface of thylakoids in a precursor form with a short C-terminal extension consisting of 8 -16 amino acids (4 -6). This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7-10). In eukaryotic organisms, the enzyme is nuclear encoded (8 -10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e. the manganese cluster in PSII (12-15). However, the absence of this extension by itself at least under the conditions exam...

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Section: Discussionmentioning

confidence: 99%

Section: Cleavage Of C-terminal Oligopeptides By Ctpa Expressed In Ementioning

confidence: 99%

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Yamamoto

Inagaki

Satoh

2001

Journal of Biological Chemistry

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“…1 and 2). Except for the SPase found in the endoplasmic reticulum, the SPases are proteases that use a serine-lysine catalytic dyad mechanism (3)(4)(5)(6)(7)(8)(9). The active site serine residue, Ser-90 in Escherichia coli SPase, is conserved in the homologous subunit within the endoplasmic reticulum signal peptidase complex.…”

mentioning

confidence: 99%

The Identification of Residues That Control Signal Peptidase Cleavage Fidelity and Substrate Specificity

Karla

Lively

Paetzel

et al. 2005

Journal of Biological Chemistry

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Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at ؊1 (P1) and small or larger aliphatic residues at the ؊3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used sitedirected mutagenesis, mass spectrometric analysis, and in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile-144 residue. Changes of the Ile-144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the ؊1 residue in a double mutant in which both Ile-86 and Ile-144 were changed to an alanine. Also, alteration of the Ile-144 and Ile-86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage following a ؊1 Asn residue. This work shows that Ile-144 and Ile-86 contribute to the signal peptidase substrate specificity and that Ile-144 is important for the accuracy of the cleavage reaction.In all living cells, proteins that are exported to the cell surface are synthesized with an N-terminal, cleavable signal peptide. The signal peptides are then proteolytically removed by a type I signal peptidase (SPase) 1 that has its catalytic domain on the trans side of the membrane.SPases have been identified and characterized in a wide variety of Gram-negative and Gram-positive bacteria, mitochondria, and the endoplasmic reticulum (for reviews, see Refs. 1 and 2). Except for the SPase found in the endoplasmic reticulum, the SPases are proteases that use a serine-lysine catalytic dyad mechanism (3-9). The active site serine residue, Ser-90 in Escherichia coli SPase, is conserved in the homologous subunit within the endoplasmic reticulum signal peptidase complex. However, it differs from the bacterial and organellar SPases by the substitution of a histidine in place of the catalytic lysine. The endoplasmic reticulum signal peptidase uses either a serine-histidine dyad mechanism or the classical serine-histidine-aspartic acid triad mechanism (4). The crystal structure of the E. coli signal peptidase with covalently attached inhibitor proved that SPase is a serine protease and is consistent with Lys-145 being involved in the activation of the nucleophilic Ser-90 (10). The structure provided a picture of how SPase can bind and cleave signal peptides from exported proteins. Fig. 1 shows a structural model of the E. coli SPase (shown in a light gray ribbon ...

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“…This penem derivative has been already shown to inhibit SPase activity of E. coli LepB (Black & Bruton, 1998), Staphylococcus aureus SpsB (Paetzel et al, 2000), Streptomyces lividans SipW, SipX, SipY and SipZ (Geukens et al, 2002), and the SPases from cyanobacterium and chloroplast thylakoids (Barbrook et al, 1996). Recently, lipopeptides have been developed which are more effective in SPase inhibition (Paetzel et al, 2002;Bruton et al, 2003).…”

Section: Transcriptional Analysis Of the L Pneumophila Lepb Genementioning

confidence: 99%

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila

et al. 2004

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Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases. INTRODUCTIONLegionella pneumophila, the causative agent of Legionnaires' disease in man, is a Gram-negative facultative intracellular parasite of monocytes and alveolar macrophages. In the environment, L. pneumophila inhabits fresh water, where it can reside and proliferate within various free-living protozoa, or is present in a biofilm-associated state. Human infection can occur following inhalation of small aerosolized droplets containing Legionella cells. Since the initial isolation of L. pneumophila in 1976, significant progress has been made in identifying virulence determinants for entry and intracellular multiplication in eukaryotic hosts. As in other Gram-negative pathogens, these virulence factors are usually proteins and are generally either secreted into the extracellular environment or attached to the cell surface (Finlay & Falkow, 1997). Although the secreted proteins are numerous and diverse, only a few pathways exist by which these proteins are transported from the bacterial cytoplasm to the extracellular environment (reviewed by Lee & Schneewind, 2001). For L. pneumophila, the presence of type II and type IV secretion systems has been described. The first type IV apparatus, encoded by the icm/dot genes , was sh...

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Inhibition by penem of processing peptidases from cyanobacteria and chloroplast thylakoids

Cited by 17 publications

References 23 publications

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

The Identification of Residues That Control Signal Peptidase Cleavage Fidelity and Substrate Specificity

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila

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