Antoine Puigserver

Sequence analysis of the aminoacylase-1 family. A new proposed signature for metalloexopeptidases.

The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease.

Canavan disease is an autosomal‐recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N‐acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K m and k cat of 0.20 ± 0.03 mM and 14.22 ± 0.48 s−1, respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H21xxE24(91aa)H116. We further investigated the active site of hASP by performing modelling studies and site‐directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc‐binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild‐type and Glu178Asp have the same K m but different k cat values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.

Specific enhancement of acylase I and acylpeptide hydrolase activities by the corresponding N-acetylated substrates in primary rat hepatocyte cultures

The specific effects of N‐acetyl‐L‐methionine on acylase I activity and of both N‐acetyl‐L‐alanyl‐L‐alanine and N‐acetyl‐L‐methionyl‐L‐alanine on N‐acylpeptide hydrolase activity were investigated in primary rat hepatocyte cultures. Each of the above two substrates is known to be much more rapidly hydrolyzed than other derivatives of the same type under optimum enzyme assay conditions. After a two‐day incubation of the substrates in the presence of primary rat hepatocyte cultures, the N‐acetylaminoacid was found to specifically induce an increase in the acylase I activity, whereas the two N‐acetylated peptides increased the acylpeptide hydrolase activity in the soluble 100000 × g fraction from the culture medium. No change in any of the enzyme activities could be detected during the same period of time when the medium was not supplemented with N‐acetylated substrates. In addition, the acylase I activity showed a dose dependent response when the N‐acetyl‐L‐methionine concentration increased from 10 fold to 50 fold. It is therefore suggested that the efficient hydrolysis of each type of substrate that occured in the 48 h hepatocyte cell cultures was due to the increase observed in the overall activity of the corresponding enzymes. The ratio of acylpeptide hydrolase to that of acylase I increased considerably when the medium was supplemented with N‐acetylpeptides, and decreased in the presence of the N‐acetylaminoacid.

Structural Properties of Porcine Intestine Acylpeptide Hydrolase

The acylpeptide hydrolase of porcine intestinal mucosa (pi-APH) is a serine peptidase belonging to the prolyl oligopeptiase family. The enzyme catalyzes the release of N-terminal acylamino acids, especially acetylamino acids, from acetylpeptides. pi-APH is an homotetramer of approximately 300 kDa. We report the loss of the native tetrameric structure of pi-APH upon citraconylation and the process was reversed at acidic pH, indicating that the subunits were noncovalently bound. Determination of free cysteines in combination with peptide mapping suggested the involvement of all cysteines in disulfide bridges. Two structural domains were identified based on the three-dimensional model of pi-APH monomer: a β-propeller fold in the N-terminal sequence (113–455) and an α/β hydrolase fold corresponding to the C-terminal catalytic domain (469–732). Preferential cleavage sites for limited proteolysis with trypsin occurred within the β-propeller domain, in agreement with the three-dimensional model. The putative role of this domain in the specificity mechanism of APH enzymes is also discussed.

Bovine Pancreatic Preproelastases I and II: Comparison of Nucleotide and Amino Acid Sequences and Tissue Specific Expression

Clones encoding bovine preproelastases I and II were isolated from a pancreatic cDNA library and were sequenced in order to define the structural characteristics of these enzymes. The bovine 947- and 884-nucleotide preproelastase I and II cDNAs encode proteins containing a signal peptide of the same length (16 amino acids), but with a slightly different number of amino acids for the activation peptide (10 and 12, respectively) and the mature enzyme (240 and 241, respectively). Considering amino acid sequences, each enzyme shares a high degree of identity (76-86%) within species. In contrast, only 55.3% identity is found between bovine elastases I and II. This difference could explain partly their own specificity. Analysis of the expression of the elastases in various bovine tissues demonstrated that they are specifically expressed in high levels in the pancreatic gland. These two approaches (structure and expression) allowed us to characterize the bovine pancreatic elastases I and II.

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.

Activation of bile salt dependent lipase by (lyso)phosphatidic acid and platelet activating factor

The activity of breast milk BSDL was assayed with or without phospholipids as extra‐intestinal effector candidates. Phosphatidic acid, lysophosphatidic acid and platelet activating factor but not phosphatidylcholine and lysophosphatidylcholine stimulated BSDL activity at least as efficiently as taurocholate. The apparent dissociation constants of PA and LPA at saturating concentrations of three different substrates were between 0.1 and 13.4 μM and that of PAF was below or equal to 200 pM. Kinetic data suggested the existence of at least one binding site for each of these effectors. PA, LPA and PAF are likely extra‐intestinal modulators of BSDL activity.