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PubMed1963

A histochemical enzyme kinetic system applied to the trypsin-like amidase and esterase activity in human mast cells.

A method for the determination of enzyme kinetic constants V(m), K(m), and K(i) in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.

Amidohydrolases↗
PubMed1998

Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity.

The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.

Amidohydrolases↗
PubMed1987

Glutamine degradation through the omega-amidase pathway in Saccharomyces cerevisiae.

A glutamine transaminase activity has been identified in Saccharomyces cerevisiae, and the existence of the omega-amidase activity previously described in this yeast has been confirmed. The glutamine transaminase utilizes different 2-oxo acids as substrates, including pyruvate and glyoxylate, and is regulated by the available nitrogen source. The glutamine transaminase activity decreases when lysine or glycine is added to the medium; the inhibition by lysine diminishes under microaerophilic culture conditions.

Amidohydrolases↗
PubMed2002

Crystallization and preliminary X-ray data of the recombinant peptide amidase from Stenotrophomonas maltophilia.

The peptide amidase from Stenotrophomonas maltophilia selectively hydrolyses the C-terminal amide bond in peptide amides. Crystals have been obtained by sitting-drop vapour diffusion from solution containing polyethylene glycol (PEG) 6000, HEPES pH 7.5, glycerine and sodium azide (NaN(3)). The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.18, b = 62.60, c = 101.91 A, beta = 90 degrees. X-ray data from these crystals diffracted at the European Synchrotron Radiation Facility (ESRF, France) ID14-1 beamline to 1.4 A.

Amidohydrolases↗
PubMed1999

Crystallization and preliminary X-ray analysis of a new L-aminopeptidase-D-amidase/D-esterase activated by a Gly-Ser peptide bond hydrolysis.

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group presumably used as a general base in the catalytic mechanism. Two crystal forms were obtained at 294 K in 13-16% PEG 2000 mono-methylether at pH 9.0, adding either 0.2 M magnesium chloride or 1 M lithium chloride. Crystals of the first form belong to the space group C2221 and diffract to 3.0 A resolution, whereas crystals of the second form belong to the space group P21212 and diffract to 2.3 A resolution. Initial screening for heavy-atom derivatives on form II crystals, has led to a well substituted Hg derivative.

Aminopeptidases↗
PubMed1993

Bacillus subtilis mutant deficient in the major autolytic amidase and glucosaminidase is impaired in motility.

The purified autolytic endo-beta-N-acetylglucosaminidase of Bacillus subtilis AC327 was cleaved with cyanogen bromide, and the N-terminal amino acid sequence of one of the peptide fragments was determined. Then, a DNA fragment containing a part of the glucosaminidase gene was cloned into Escherichia coli JM109 using synthetic oligonucleotides as probes whose sequences had been deduced from the N-terminal amino acid sequence. Zymographic analysis showed that the resultant glucosaminidase-deficient strain lacked a 35-kDa lytic band in addition to a 90-kDa lytic one corresponding to the glucosaminidase. A double mutant strain deficient in the major two autolysins (amidase and glucosaminidase) exhibited greatly impaired motility on a swarm plate whereas the single mutant strains were motile.

Acetylglucosaminidase↗
PubMed2000

Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles.

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9.

Acrylic Resins↗
PubMed1979

Release of soluble peptidoglycan from growing gonococci: hexaminidase and amidase activities.

Peptidoglycan (PG) turnover in exponentially growing Neisseria gonorrhoeae RD5 type 4 was accompanied by release of soluble PG fragments into the medium. Turnover of the D-[14C]glucosamine-labeled glycan moiety and of the meso-[3H]diaminopimelic acid (DAP)-labeled peptide region occurred at similar rates (ca. 35% per generation). Turnover of D-[14C]alanine-labeled sites within the peptide side chain of PG occurred at roughly twice this rate; no turnover of L-[3H]proline-labeled protein was detected. Gel filtration of supernatants of cultures grown in the presence of labeled DAP, glucosamine, and D-alanine as described above and paper chromatography of hydrolyzed peak fractions revealed four major types of soluble PG. Two of these contained both peptide and glycan moieties and appeared to represent forms of disaccharide peptide monomers and dimers. The other two were (i) a 3H-labeled product lacking 14C and (ii) a 14C-containing product lacking 3H, which were similar in size to that expected for free tetrapeptides and free disaccharides, respectively. Together the appearance of these PG fragments and the concurrent turnover of glycan and peptide regions indicate that both glycan splitting and amidase PG hydrolase activities are involved in the turnover of PG in growing gonococci. If released during gonococcal infections, similar soluble PG fragments might influence the consequences of host-gonococcus interactions.

Amidohydrolases↗
PubMed1991

Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides.

Proteolytic enzymes from the organism Porphyromonas gingivalis are believed to be involved in the development of periodontitis. Studies on both crude extracts and purified trypsinlike enzymes from this organism indicate that substantial stimulation of both amidase and proteinase activities can be obtained during incubation with glycine-containing compounds. We postulate that P. gingivalis may have developed this unusual property to take advantage of the glycine-rich environment which occurs during the periodontitis-associated degradation of gingival collagen. The finding of such a stimulation in crevicular fluids from discrete periodontal sites has been correlated with the presence of P. gingivalis and could be utilized for the early detection of infection by this organism during the onset of periodontitis.

Amidohydrolases↗
PubMed2005

Peptidoglycan recognition protein 2 (N-acetylmuramoyl-L-Ala amidase) is induced in keratinocytes by bacteria through the p38 kinase pathway.

Human peptidoglycan recognition protein 2 (PGLYRP2) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and is constitutively produced in the liver and secreted into the blood. Here we demonstrate that PGLYRP2 was not expressed in healthy human skin and had low expression in the eye. However, upon exposure to gram-positive and gram-negative bacteria or cytokines, PGLYRP2 expression was highly induced in keratinocytes and to a lower level in corneal epithelial cells. Expression of PGLYRP2 was not induced in nonepithelial cells. Exposure of keratinocytes to bacteria induced keratinocyte differentiation and stress response and inhibited activation of signal transduction molecules involved in cell proliferation. Induction of PGLYRP2 expression correlated with expression of differentiation markers (cytokeratins and transglutaminase). Bacteria induced activation of p38 mitogen-activated protein kinase (MAPK) in keratinocytes, which was required for the induction of PGLYRP2 expression, because induction of PGLYRP2 transcription by bacteria was inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by a dominant-negative p38 construct. Induction of PGLYRP2 expression by bacteria (in contrast to expression of human beta-defensin-2) was not mediated by Toll-like receptor 2 or 4. PGLYRP2 may function in the skin and the eyes as an inducible scavenger of proinflammatory peptidoglycan.

Bacterial Physiological Phenomena↗
PubMed1995

Transcriptional analysis of the amidase operon from Pseudomonas aeruginosa.

The transcriptional start point for the amidase structural gene (amiE) of Pseudomonas aeruginosa has been identified, and the promoter (pE) has been shown to function constitutively, as predicted for a system regulated by transcription antitermination. Northern (RNA) analysis results show that in cells grown under inducing conditions, a major 1.3-kb amiE transcript arises from pE, and in addition, a larger transcript of approximately 5.0 kb in length has been shown to derive from the same promoter, encoding all of the genes of the operon. DNA sequencing and S1 nuclease mapping have located a transcription terminator downstream of amiE, which terminates approximately half of the pE transcripts. Previously, two RpoN-dependent promoter-like sequences (pN1 and pN2) were identified upstream of the negative regulator gene, amiC, and we have now constructed a promoter probe vector which shows weak constitutive promoter activity within this region. This promoter would be expected to provide basal levels of expression of the amiC and amiR regulatory genes to allow induction of the system.

Amidohydrolases↗
PubMed1995

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Acetylglucosaminidase↗
PubMed2003

Identification of MpaA, an amidase in Escherichia coli that hydrolyzes the gamma-D-glutamyl-meso-diaminopimelate bond in murein peptides.

MpaA amidase was identified in Escherichia coli by its amino acid sequence homology with the ENP1 endopeptidase from Bacillus sphaericus. The enzymatic activity of MpaA, i.e., hydrolysis of the gamma-D-glutamyl-diaminopimelic acid bond in the murein tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelic acid, was demonstrated in the cell extract of a strain expressing mpaA from a multicopy plasmid. An mpaA mpl (murein peptide ligase) double mutant accumulated large amounts of murein tripeptide in its cytoplasm, consistent with the premise that MpaA degrades the tripeptide if its recycling via the peptidoglycan biosynthetic pathway is blocked.

Amidohydrolases↗
PubMed1985

An improved method of ammonia determination, applicable to amidases and other ammonia-producing enzyme systems of mycobacteria.

The colorimetric estimation of amidase activity, using both qualitative and quantitative determinations of ammonia, is widely used for the differentiation of mycobacteria. At present the generally used phenol-hypochlorite method requires heating of the test solution to 90 degrees C for 30 min or to boiling for 5 min. At room temperature at least 2 h are necessary to obtain a full and stable color. Heating is also disadvantageous because it increases the vaporization of toxic phenol vapors and it may lead to the formation of insoluble manganese dioxide, which interacts with the photometric determination. We found that the addition of ketones (preferably acetone) to the catalyst solution (MnSO4) accelerates the reaction in such a manner that heating is not necessary and the full color development can be obtained within 6 min. The proposed method is superior to the conventional ones because (1) the fully developed color can be obtained after 6 minutes without heating; (2) boiling, which increases the volatilization of phenol and creates dangers for the laboratory staff and the equipment, can now be reduced; and (3) the formation of manganese dioxide in the test solution is avoided.

Amides↗
PubMed2006

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium.

BACKGROUND: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of beta-lactam antibiotics were systematically improved. RESULTS: For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. CONCLUSION: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

Journal Article↗
PubMed1996

Clinical application of basic arginine amidase in human male urine.

Basic human urinary arginine amidase (or esterase, called BHUAE) which is only found in male urine, was measured from normal volunteers between the age of 4 and 70 years using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate. BHUAE increases during early adolescence, between 8 to 17 years of age. Then, BHUAE decreases in the twenties and takes a certain range of value in the mature age group, between the late thirties and fifties. In patients with prostate cancer, a significant increase in BHUAE was demonstrated in comparison with the healthy male group (control) over 55 years old. On the other hand, patients with benign prostatic hypertrophy showed no significant elevation of this enzyme activity. It would appear that the measurement of BHUAE in urine can be used as a marker of prostate cancer in an advanced age group.

Adolescent↗
PubMed1996

Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids.

Mouse brain microsomes contain an amidase that catalyzes the hydrolysis of N-arachidonylethanolamide to arachidonic acid and ethanolamine. The enzymatic activity is dependent on the protein concentration of the microsomes and observed over a wide range of pH, 7.4 to 9.5. Kinetic analysis indicated that K(m) (microM) and Vmax (nmol/min/mg protein) were 17.7 +/- 4.1 and 1.81 +/- 0.32, respectively. Cannabidiol (CBD), cannabinol (CBN) and delta 9-tetrahydrocannabinol (delta 9-THC) significantly inhibited the hydrolysis of the amide by mouse brain microsomes. At a concentration of 160 microM, the inhibitory potency decreased in the following order, CBD (66%) > CBN (46%) > delta 9-THC (31%).

Amidohydrolases↗