Acetylcholinesterase inhibitor SDZ ENA 713 (Rivastigmine) increases brain pyrrolidone carboxyl peptidase activity.

Pyroglutamyl-ended forms of amyloid-beta-peptide are present in senile plaques in some individuals with Alzheimer type dementia. Single oral administration of the acetylcholinesterase inhibitor SDZ ENA 713 (rivastigmine (+)-(S)-N-ethyl-3-[(1-dimethylamino)ethyl]-N-methylphenylcarbamate hydrogen tartrate) increases basal and K(+)-stimulated pyrrolidone carboxyl peptidase (Pcp) activity in mice frontal cortex synaptosomes in a dose-dependent manner. These results suggest that this drug may ameliorate ATD cognitive deficits acting not only facilitating cholinergic transmission but also avoiding the formation of pyroglutamyl-ended amyloid-beta-peptides (A beta pE) deposition through the activation of Pcp.

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity.

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E.

The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi.

Modes of action of five different endopectate lyases from Erwinia chrysanthemi 3937.

Five endopectate lyases from the phytopathogenic bacterium Erwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character.

Synthesis of the two monomethyl esters of the disaccharide 4-O-alpha-D-galacturonosyl-D-galacturonic acid and of precursors for the preparation of higher oligomers methyl uronated in definite sequences.

Methyl (alpha-D-galactopyranosyluronic acid)-(1-->4)-D-galactopyranuronate and methyl alpha-D-galactopyranosyl-uronate-(1-->4)-D-galactopyranuronic acid have been synthesized by coupling methyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (3) or benzyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (4) with benzyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate and methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate, respectively, using N-iodosuccinimide and trifluoromethanesulphonic acid as promoters, followed by removal of the benzyl groups. The 4'-OH unprotected dimers benzyl (methyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate and methyl (benzyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate were prepared from methyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and benzyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and acceptors 4 or 3, respectively. These compounds have been designed to serve as precursors for the preparation of higher-membered D-galacturonic acid oligomers methyl esterified in definite positions.

Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains.

Biochemical characterization of the pectate lyase PelZ of Erwinia chrysanthemi 3937.

To degrade the plant pectin, the phytopathogenic bacterium Erwinia chrysanthemi produces a set of at least seven endo-pectate lyases (Pels). Five major (PelA, PelB, PelC, PelD and PelE) and two minor isoenzymes (PelL and PelZ) have been identified. PelZ is an extracellular enzyme secreted by the Out system.

Antagonistic effect of CRP and KdgR in the transcription control of the Erwinia chrysanthemi pectinolysis genes.

The main virulence factors of the phytopathogenic bacteria Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. The cyclic AMP receptor protein (CRP) was identified as the main activator of the pectinolysis genes. Gel shift and DNase I footprinting experiments showed that the purified E.

Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937.

Pyroglutamic acid and iron regulate the expression of the pcp gene in Pseudomonas fluorescens MFO.

Pyrrolidone carboxyl peptidase (Pcp) is an aminopeptidase (EC 3.4.11.

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi.

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS.

Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins.

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS.

The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi.

The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. Although physiological studies suggested that pectinase production in Erwinia species is subjected to catabolite repression, the direct implication of the cyclic AMP receptor protein (CRP) in this regulation has never been demonstrated. To investigate the role of CRP in pectin catabolism, we cloned the E.

Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors.

In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE. Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes.

Regulation of pelZ, a gene of the pelB-pelC cluster encoding a new pectate lyase of Erwinia chrysanthemi 3937.

The phytopathogenic enterobacterium Erwinia chrysanthemi 3937 produces five major and several secondary endo-pectate lyases encoded by the pel genes. Most of these genes are arranged in clusters on the bacterial chromosome. The genomic region surrounding the pelB-pelC cluster was supposed to be involved in the regulation of PelB and PelC synthesis.

Bacterial aminopeptidases: properties and functions.

Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasic activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains.

Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase.

On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments. Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the synthetic chromogenic substrate pyroglutamyl-beta-naphthylamide and the dipeptide pyroglutamyl-alanine. Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp.

Purification and functional characterization of PecS, a regulator of virulence-factor synthesis in Erwinia chrysanthemi.

The Erwinia chrysanthemi pecS gene encodes a repressor that negatively regulates the expression of virulence factors such as pectinases or cellulases. The cloned pecS gene was overexpressed using a phage T7 system. The purification of PecS involved DEAE-anion exchange and TSK-heparin columns and delivered the PecS protein that was purified to homogeneity.

The Erwinia chrysanthemi pecT gene regulates pectinase gene expression.

A new type of Erwinia chrysanthemi mutant displaying a derepressed synthesis of pectate lyase was isolated. The gene mutated in these strains, pecT, encodes a 316-amino-acid protein with a size of 34,761 Da that belongs to the LysR family of transcriptional activators and presents 61% identity with the E. coli protein LrhA.

Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937.

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library.

Purification and characterization of the nuclease NucM of Erwinia chrysanthemi.

The major periplasmic nuclease of Erwinia chrysanthemi strain 3937, NucM, has been purified near to homogeneity by a one step purification procedure, using chromatography on a sulfopropyl column. NucM cleaves randomly single and double-stranded DNA and RNA. It does not need divalent cations for its action, and is more active in low salt buffers.

Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes.

Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi.

An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein.

The structure of Bacillus subtilis pectate lyase in complex with calcium.

We have solved the structure of the Bacillus subtilis pectate lyase (BsPel) in complex with calcium. The structure consists of a parallel beta-helix domain and a loop region. The alpha L-bounded beta-strand seen in BsPel is a new element of protein structure and its frequent occurrence suggests it is an important characteristic of the parallel beta-helix.

Pyrrolidone carboxyl peptidase (Pcp): an enzyme that removes pyroglutamic acid (pGlu) from pGlu-peptides and pGlu-proteins.

Pyrrolidone carboxyl peptidase (EC 3.4.11.