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PubMed1996

Production of R-(-)-Ketoprofen from an Amide Compound by Comamonas acidovorans KPO-2771-4.

R-(-)-2-(3(prm1)-Benzoylphenyl)propionic acid [R-(-)-ketoprofen] was produced from racemic 2-(3(prm1)-benzoylphenyl)propionamide (keto-amide) by the isolated bacterial strain Comamonas acidovorans KPO-2771-4. Sodium fumarate as the carbon source and 2-azacyclononanone or isobutyronitrile as the enhancer in the culture medium were effective for bacterial growth and the enhancement of R-(-)-ketoprofen-producing activity. R-(-)-Ketoprofen produced from the keto-amide by resting cells was present in 99% enantiomeric exess. C. acidovorans KPO-2771-4 has an R-enantioselective amidase for keto-amide because the purified amidase from the bacterium hydrolyzed keto-amide, producing optically pure R-ketoprofen and ammonia.

Journal Article↗
PubMed2006

Identification of antigenic components of Staphylococcus epidermidis expressed during human infection.

A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease.

Amino Acid Sequence↗
PubMed1980

Release of soluble peptidoglycan from growing conococci: demonstration of anhydro-muramyl-containing fragments.

Previous analysis of soluble peptidoglycan (PG) fragments released by exponentially growing gonococci implicated the combined action of both hexosaminidase and amidase activities in PG turnover. Current studies further characterized PG fragments which were labeled in the glycan with D-glucosamine and in the peptide moiety with meso-diaminopimelic acid of L- and D-alanine. Labeled PG fragments were isolated by gel filtration and characterized on the bases of (i) KD values, (ii) free amino group analysis using fluorodinitrobenzene, (iii) borohydride reduction, (iv) alkali-catalyzed beta-elimination, (v) paper chromatography in various solvents, (vi) electrophoretic mobility at various pH values, (vii) digestibility by Charonia lampas glycosidases, and (viii) content of labeled D- and L-alanine. A set of well-characterized PG fragments was used as standards. The monomer fraction (the major extracellular product) was found to contain two components. Most (about 80%) appeared to be N-acetylglucosaminyl-beta-1 leads to 4-1,6-anhydro-N-acetylmuramyl-L-ala-D-glu-meso-diaminopimelic acid; the remainder was the corresponding disaccharide tetrapeptide containing a C-terminal D-alanine. An unusual feature of these products was the presence of the anhydro-muramyl (non-reducing) ends, reflecting the activity of a gonococcal transglycosylase, and the near absence of products containing detectable reducing ends. Otherwise, the structures of the monomer fragments were typical of those expected for a gram-negative bacterium (chemotype I). The corresponding peptide-cross-linked dimer and the free disaccharide also contained nonreducing ends, exclusively. Free peptides (products of amidase activity) consisted of both tripeptide and tetrapeptide. In summary, all gonococci examined appear to possess an unusual transglycosylase activity which contributes to the release of soluble PG fragments containing nonreducing, anhydro-muramyl ends. The release of these fragments in vivo might be a unique aspect of gonococci-host interactions.

Amino Acids↗
PubMed1992

Role of the bacterial cell wall in middle ear inflammation caused by Streptococcus pneumoniae.

The pathogenesis of middle ear inflammation caused by Streptococcus pneumoniae was explored in the chinchilla model with different pneumococcal cell wall (CW) preparations, including isolated native CW, M1 muramidase CW (M1-CW) digest, amidase CW digest, and M1 peptidoglycan (M1-PG) digest. Inflammatory cell and lysozyme concentrations in middle ear fluid (MEF) were measured between 6 and 72 h after the middle ears were inoculated with one of the preparations or sterile saline. Middle ear histopathology was measured quantitatively at 72 h. Native CW, M1-CW digest, and amidase-CW digest caused significantly more inflammatory cell influx and lysozyme accumulation in MEF than saline did. M1-PG digest also caused more inflammatory cell influx and lysozyme accumulation in MEF than saline did but caused less inflammation than native CW or either CW digest. Epithelial metaplasia was significantly greater in ears inoculated with native CW than in ears inoculated with the CW or PG digest or with saline. Pneumococcal CW is, therefore, the principal factor that initiates middle ear inflammation in acute pneumococcal otitis media, and CW teichoication seems to be important in initiating this response.

Amidohydrolases↗
PubMed2004

The Burkholderia cepacia epidemic strain marker is part of a novel genomic island encoding both virulence and metabolism-associated genes in Burkholderia cenocepacia.

The Burkholderia cepacia epidemic strain marker (BCESM) is a useful epidemiological marker for virulent B. cenocepacia strains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): an N-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66 family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated the B. cenocepacia island (cci), is the first genomic island to be defined in the B. cepacia complex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.

Amidohydrolases↗
PubMed1974

Effects of ammonium, L-glutamate, and L-glutamine on nitrogen catabolism in Aspergillus nidulans.

During growth of Aspergillus nidulans in medium containing ammonium the specific activities of most enzymes involved in catabolism of nitrogen sources are low (ammonium repression). The gdhA10 lesion, which results in loss of nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase activity, has been shown to lead to partial relief of ammonium repression of three amidase enzymes as well as histidase. The areA102 lesion led to altered levels of these enzymes but did not greatly affect ammonium repression. The double mutant areA102,gdhA10 was almost completely insensitive to ammonium repression of two of the amidase enzymes and histidase. This suggests that an interaction between the areA and gdhA genes in determining responses to ammonium occurs. Growth of mycelium in medium containing l-glutamate has been found to result in lowered levels of all four enzymes, and this occurs in strains insensitive to ammonium repression. Very strong repression in all strains occurred during growth in medium containing l-glutamine. Relief of these repressive effects of glutamate and glutamine was blocked by cycloheximide. Glutamate and glutamine had similar effects on the production of extracellular protease activity, and growth on glutamine led to low levels of urate oxidase. In contrast to the above enzymes, nitrate reductase was insensitive to the effects of glutamine and glutamate, even though this enzyme is very sensitive to ammonium repression. Although other possibilities exist, it is suggested that there may be mechanisms of general control of nitrogen-catabolic enzymes other than ammonium repression.

Amidohydrolases↗
PubMed1976

Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells.

The specific activities of three murein hydrolases, carboxypeptidase I, carboxypeptidase II, and amidase were studied with respect to cell division in toluene-treated cells of Escherichia coli K-12. Carboxypeptidase I and amidase activities were constant throughout the division cycle in cells of D11/lac+pro+. Detectable carboxypeptidase II activity varied and was highest at the time of division by a factor of three. Carboxypeptidase II specific activity was also correlated with cell division in BUG 6, a temperature-sensitive mutant (J.N Reeve, D.J. Groves, and D.J. Clark, 1970). Fifteen minutes after shifting BUG 6 from 42 C (nondividing conditions) to 32 C (dividing conditions), there was a rapid resumption of cell division, accompanied by a 10-fold increase in the specific activity of carboxypeptidase II. These results demonstrate a correlation between detectable carboxypeptidase II activity and cell division as reflected by activity in toluene-treated cells. The subcellular location of carboxypeptidase II, a soluble enzyme was found to be periplasmic since it was released by tris(hydroxymethyl)-aminomethane-ethylenediaminetetraacetate treatment and osmotic shock, two methods known to release periplasmic enzymes.

Amidohydrolases↗
PubMed2000

N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.

Acetylation↗
PubMed2002

Expression of Mycobacteriophage Ms6 lysis genes is driven by two sigma(70)-like promoters and is dependent on a transcription termination signal present in the leader RNA.

A mycobacteriophage Ms6 strong promoter region (P(lys)) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem sigma(70)-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P(lys) drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that beta-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

Amino Acid Sequence↗
PubMed2005

Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP.

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Allophanate Hydrolase↗
PubMed1969

Mechanism of autolysis of isolated cell walls of Staphylococcus aureus.

Autolysis of isolated cell walls of Staphylococcus aureus strain Copenhagen was accompanied by the release of 1 mole of N-terminal alanine per mole of glutamic acid. No other N-terminal amino acids and no C-terminal amino acids were released. These observations indicated that complete hydrolysis of N-acetylmuramyl-l-alanine linkages ("amidase" action) had occurred. This was confirmed by fractionation and analysis of the products. Hydrolysis of 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages also occurred to a variable extent; on one occasion, complete degradation to disaccharides and hexosamine-free polypeptides (with intact pentaglycine cross-bridges) occurred. In one other instance, hydrolysis within pentaglycine bridges also occurred. Analyses of intact cell walls indicated that, in vivo, glycine endopeptidase activity was negligible and amidase activity was low, but that endo-beta-N-acetylglucosaminidase hydrolysed about 8% of the N-acetylglucosaminyl-N-acetylmuramic acid linkages. Autolysis of isolated cell walls was too slow for the enzymes isolated with them to have significant action during this isolation. The possible functions of these autolytic activities are discussed.

Alanine↗
PubMed1995

Identification and functional importance of plasma kallikrein in the synovial fluids of patients with rheumatoid, psoriatic, and osteoarthritis.

OBJECTIVES: To determine and identify, unequivocally, if plasma kallikrein (PK) is present in the synovial fluid of patients with rheumatoid (RA), psoriatic (PA) and osteo (OA) arthritis, and to consider its functional importance in the inflamed joint. METHODS: Therapeutically aspirated synovial fluids (pooled and individual samples, n = 66) were obtained from patients with arthritis. In addition, serum (n = 14) was collected from RA patients, and saliva (n = 10) and urine (n = 10) from normal individuals. Enzymic (amidase) and immunoreactive activities of PK and its precursor, prokallikrein (PPK), were determined. The presence of PK was assessed by incubation with soya bean trypsin inhibitor (SBTI), and by adsorption with anti-PK antibody linked to Sepharose. An enzyme-linked immunosorbant assay (ELISA) for PK was developed for quantitative measurement of total PK in biological fluids. Enhancement of the PK dose-response by RA synovial fluid made it necessary to remove RF from synovial fluids before determination of PK by ELISA. RESULTS: Amidase activity was demonstrated in synovial fluid pools and shown to be inhibited completely by SBTI, and removed by prior treatment with anti-PK Sepharose. Total PK activity (PK + PPK) from individual synovial fluid specimens did not differ significantly between patients with RA (median activity 76 mU/g protein), PA (80 mU/g protein) or OA (60 mU/g protein). Similar results were obtained when active PK alone was measured. No correlation was found between active PK or total PK values and the severity score for individual joints. Most of the measured immunoreactivity was removed by adsorption with anti-PK antibody linked to Sepharose. CONCLUSION: The results support the hypothesis that plasma kallikrein is present in synovial fluid. The enzyme may be important in the pathogenesis of inflamed joints.

Adult↗
PubMed1983

Isoniazid-induced hepatic steatosis in rabbits: an explanation for susceptibility and its antagonism by pyridoxine hydrochloride.

Steatosis was induced in rabbits by subacute administration of isoniazid (INH, 50 mg/kg po). Concomitant treatment with pyridoxine (vitamin B6, 25 mg/kg po) antagonized both development of the hepatic lesions and the elevation of plasma concentrations of lipids. Rabbit acetylating ability was sixfold that of male Wistar rats, a species susceptible to hepatic cell necrosis, whereas hepatic cytochrome P-450 and NADPH-cytochrome c reductase were significantly lower than that observed in control or phenobarbital-induced rats. Examination of the hepatic hydrolysis of the amide bonds of INH and acetylisoniazid (AcINH) indicated that the isonicotinoyl bond of AcINH was the bond most susceptible to amidase hydrolysis in both species; but rabbits possessed the greater amidase activity: 5- to 20-fold greater than control rats and 2- to 7-fold greater than the phenobarbital-induced rats. Consequently, INH-induced hepatic fatty degeneration in rabbits was attributed to increased hepatic exposure to INH-derived primary amine functional groups, and its antagonism by vitamin B6 was attributed to the deactivation of the primary amine by pyridoxal hydrazone formation.

Animals↗
PubMed1971

The separation of alpha-2 macroglobulin into five components with differing electrophoretic and enzyme-binding properties.

The alpha-2 macroglobulins from human serum and plasma were isolated by Bio-Gel P-300 and A5m gel filtration. The material showed a single peak on sedimentation velocity ultracentrifugation, a mol wt of 650,000 by sedimentation equilibrium ultracentrifugation, and a major precipitin arc in the alpha-2 macroglobulin region by immunoelectrophoresis against whole human serum. Two bands were observed in the alpha-2 macroglobulin region when acrylamide gel electrophoresis was performed with a pH 8.9 running gel. When a pH 7.8 gel was used, five electrophoretic species were observed. In both cases, the preaddition of stoichiometric amounts of trypsin or chymotrypsin added to alpha-2 macroglobulin resulted in disappearance of slower bands leaving only one band on acrylamide gel electrophoresis patterns. Preparative acrylamide gel electrophoresis separated alpha-2 macroglobulin obtained from Bio-Gel into five closely-spaced species. Separation was sufficiently adequate to show that those species of alpha-2 macroglobulin which bound trypsin and chymotrypsin were represented by slower moving species and that the fastest moving material had lost virtually all of the ability to bind these enzymes. Preparative acrylamide gel electrophoresis of a mixture of alpha-2 macroglobulin-trypsin complex and alpha-2 macroglobulin revealed that the fast moving component was alpha-2 macroglobulin-trypsin complex and that the slower moving material was unbound alpha-2 macroglobulin. The naturally occurring amidase activity of the alpha-2 macroglobulin using benzoylarginine-p-nitroanilide (BAPNA) as substrate was investigated and unlike its trypsin-binding activity, amidase activity was found to be of the same specific activity in all electrophoretic fractions. Binding of trypsin and chymotrypsin to alpha-2 macroglobulin revealed that alpha-2 macroglobulin maximally bound 2 moles of trypsin and 1 mole of chymotrypsin. When the enzymes were added simultaneously there was competition. Chymotrypsin added to alpha-2 macroglobulin before the addition of trypsin prevented all trypsin binding even though only one site was filled with chymotrypsin. These results were explained by the acrylamide gels which showed that 1 mole of chymotrypsin was sufficient to convert all the alpha-2 macroglobulin to a species with the fastest mobility which no longer binds additional enzyme.

Acrylamides↗
PubMed1997

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Amidohydrolases↗
PubMed2006

The peptidoglycan recognition proteins (PGRPs).

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules present in insects, mollusks, echinoderms, and vertebrates, but not in nematodes or plants. PGRPs have at least one carboxy-terminal PGRP domain (approximately 165 amino acids long), which is homologous to bacteriophage and bacterial type 2 amidases. Insects have up to 19 PGRPs, classified into short (S) and long (L) forms. The short forms are present in the hemolymph, cuticle, and fat-body cells, and sometimes in epidermal cells in the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes. The expression of insect PGRPs is often upregulated by exposure to bacteria. Insect PGRPs activate the Toll or immune deficiency (Imd) signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyze peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which are secreted; it is not clear whether any are directly orthologous to the insect PGRPs. One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and reduces its proinflammatory activity; PGLYRP-2 is secreted from the liver into the blood and is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs are bactericidal proteins that are secreted as disulfide-linked homo- and hetero-dimers. PGLYRP-1 is expressed primarily in polymorphonuclear leukocyte granules and PGLYRP-3 and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine. These three proteins kill bacteria by interacting with cell wall peptidoglycan, rather than permeabilizing bacterial membranes as other antibacterial peptides do. Direct bactericidal activity of these PGRPs either evolved in the vertebrate (or mammalian) lineage or is yet to be discovered in insects.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of serine carboxypeptidases from Absidia zychae.

Among 29 strains of zygomycetes screened for serine carboxypeptidases, Absidia zychae NRIC 1199 showed the highest enzyme production. Two serine carboxypeptidases, CPZ-1 and CPZ-2, were purified to a homogeneous state from an extract of koji culture of A. zychae NRIC 1199. Purified CPZ-1 and CPZ-2 showed similar properties except the isoelectric point (pI); The pI of CPZ-1 and CPZ-2 were 4.50 and 4.65, respectively. The molecular weights of the CPZ-1 and CPZ-2 were 48,000 by SDS-PAGE and gel filtration. Among the proteinase inhibitors tested, phenylmethylsulfonyl fluoride and monoiodoacetic acid strongly inhibited the enzyme activity. The optimum pHs of CPZ-1 and CPZ-2 were 4.2 towards Z-Glu-Tyr. It is shown that the substrate specificities of CPZ-1 and CPZ-2 were dependent on the presence of bulky amino acid residues in the penultimate position (P1) for the small Z-peptides. However, in spite of the presence of Gly, Asp, Arg, or Pro in the P1 position, oligopeptides were hydrolyzed rapidly. CPZ-1 and CPZ-2 had not only carboxypeptidase but also carboxyamidase and amidase activities, and acted preferentially as a carboxyamidase for C-terminal amidated peptides. The hydrophobicity of P2 and P3 positions and the bulkiness of P1 and P'1 positions of the substrate may be important for carboxyamidase and amidase activities.

Amino Acid Sequence↗