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PubMed2005

The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles.

The signature amidase from the extremophile archeum Sulfolobus solfataricus is an enantioselective enzyme that cleaves S-amides. We report here that this enzyme also converts nitriles in the corresponding organic acid, similarly to the well characterized amidase from Rhodococcus rhodochrous J1. The archaeal and rhodococcal enzymes belong to the signature amidases and contain the typical serine-glycine rich motif. They work at different optimal temperature, share a high sequence similarity and both contain an additional CX3C motif. To explain their dual specificity, we built a 3D model of the structure of the S. solfataricus enzyme, which suggests that, in addition to the classical catalytic Ser-cisSer-Lys, a putative additional Cys-cisSer-Lys catalytic site, likely to be responsible for nitrile hydrolysis, is present in these proteins. The results of random and site-directed mutagenesis experiments, as well as inhibition studies support our hypothesis.

Amides↗

PubMed1973

Mutant of Bacillus subtilis with a temperature-sensitive autolytic amidase.

A procedure incorporating a petri plate assay for autolysins has been developed for the systematic isolation of mutants with a temperature-sensitive autolytic amidase. We suggest maa as the name for the gene for this enzyme. One such mutant has been characterized as having a heat-sensitive amidase in a variety of studies with both crude autolysin preparations and purified amidase solutions.

Alanine↗

PubMed1976

Evidence for a role of N-acetylmuramyl-L-alanine amidase in septum separation in Escherichia coli.

Septum formation and septum separation have been studied in a chain-forming mutant of Escherichia coli K-12 bearing the envA mutation and its parental strain. In comparison to the wild type, the mutant showed a sixfold reduction in the specific activity of the enzyme, N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), part of which was associated to the outer membrane. Genetic as well as physiological suppression of chain formation resulted in an increase in amidase activity. The addition of N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid to growing wild-type cells and to cells bearing the envA mutation caused an inhibition of cell separation and an increased frequency of visible septa. The kinetics of septum formation and separation was followed in chains by the use of ampicillin and nalidixic acid. The latter drug inhibited initiation of new septa but allowed preformed ones to go to cell separation at a rate corresponding to that of steady-state growing cells. Ampicillin treatment, on the other hand, resulted in a more rapid decrease in the frequency of septa. The disparate effects of ampicillin and nalidixic acid were not explained by a difference in amidase activity but could be due to an inhibitory effect of ampicillin on a septal peptidoglycan fusing activity.

Amidohydrolases↗

PubMed1996

Purification and properties of an amidase from Rhodococcus erythropolis MP50 which enantioselectively hydrolyzes 2-arylpropionamides.

An enantioselective amidase from Rhodococcus erythropolis MP50 was purified to homogeneity. The enzyme has a molecular weight of about 480,000 and is composed of identical subunits with molecular weights of about 61,000. The NH2-terminal amino acid sequence was significantly different from previously published sequences of bacterial amidases. The purified amidase hydrolyzed a wide range of aliphatic and aromatic amides, The highest enzyme activities were found with amides carrying hydrophobic residues, such as pentyl or naphthoyl. The purified enzyme converted racemic 2-phenylpropionamide, naproxen amide [2-(6-methoxy-2-naphthyl) propionamide], and ketoprofen amide [2-(3'-benzoylphenyl)propionamide] to the corresponding S-acids with an enantiomeric excess of >99% and an almost 50% conversion of the racemic amides. The enzyme also hydrolyzed different alpha-amino amides but without significant enantioselectivity.

Amidohydrolases↗

PubMed1999

Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores.

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.

Amidohydrolases↗

PubMed2006

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa.

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M(r) of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.

Amidohydrolases↗

PubMed1988

The inhibition of boar acrosin amidase activity by sulfated polysaccharides.

Carbohydrate-protein interactions are known to be important in gamete interactions. We therefore investigated the inhibition of boar sperm acrosin amidase activity by carbohydrates. The sulfated polysaccharides fucoidan and dextran sulfate inhibited amide hydrolysis whereas dextran and various monosaccharides did not inhibit acrosin amidase activity. The kinetics of the inhibition corresponded to those characteristic when multiple forms of an enzyme are present. Such a kinetic result was consistent with the presence of the known autolytically produced forms of acrosin. It was previously shown that sulfated polysaccharides inhibit sperm-egg binding and that acrosin binds carbohydrate. We propose that the sulfated polysaccharide inhibition of acrosin amidase activity observed here is causally related to the previously observed sulfated polysaccharide inhibition of sperm binding to the zona pellucida.

Acrosin↗

PubMed1999

Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor.

Isoniazid (INH), a widely used drug in the prophylaxis and treatment of tuberculosis, is associated with a 1 to 2% risk of severe and potentially fatal hepatotoxicity. There is evidence that the INH metabolite hydrazine plays an important role in the mechanism of this toxicity. Metabolism of INH leads to the production of hydrazine via both direct and indirect pathways. In both cases, the activity of an INH amidase is required to hydrolyze an amide bond. In the present study, using a model of INH-induced hepatotoxicity in rabbits, pretreatment of rabbits with the amidase inhibitor bis-p-nitrophenyl phosphate 30 min before injection of INH inhibited the formation of INH-derived hydrazine and decreased measures of hepatocellular damage, hepatic triglyceride accumulation, and hypertriglyceridemia. Bis-p-nitrophenyl phosphate also potently inhibited the production of hydrazine from INH in in vitro microsomal incubations (IC50 2 microM). Although hepatic glutathione stores are decreased, they are not depleted in animals with INH-induced hepatotoxicity. Significant effects on hepatic microsomal cytochrome P-450 1A1/2 and cytochrome P-450 2E1 activities suggest that these isozymes may be involved in the mechanism of the toxicity. In conclusion, this study demonstrates the importance of amidase activity in this rabbit model of hepatotoxicity and provides additional evidence in support of the role of hydrazine in the mechanism of INH-induced hepatotoxicity.

Amidohydrolases↗

PubMed2000

[Studies of cloning and expression of rat alpha-amidase gene in Streptomyces lividans].

alpha-Amidase catalyzes the C-terminal amidation of active polypeptides in the Nerve-Endocrine system. It is important for full biological activity of the polypeptides. By using the total RNA of rat atrium as a templet, the cDNA encoding alpha-amidase was amplified by RT-PCR and sequenced. The cDNA was fused with the coding sequence of mel C1 signal peptide to produce fusion gene mel/AE for secretive expression in Streptomyces lividans. The fusion gene mel/AE was inserted into plasmid pIJ680 and the recombinant plasmid pIJ-mel/AE680 was obtained. The results of SDS-PAGE and biological activity showed that the recombinant strain S. lividans TK54[pIJ-mel/AE 680] produced secretly alpha-amidase.

Animals↗

PubMedDate not supplied

[The nature of differences in amidase and esterase activities of some acyltrypsins].

Specific trypsin substrates (esters, anilides, amides, peptides) were shown to accelerate deacetylation of monoacetylated trypsin. The amidase activity of monoacetyl-, monopropyonyl-, and tetraformyl-trypsin was not manifested if the amidase activity of native enzyme was suppressed in these preparations by the ester substrates (benzoylarginine ethyl ester or p-nitrophenyl acetate). Therefore the differences in the residual amidase and esterase activities of these acylated trypsin preparations found earlier did not contradict the universality of the acylenzyme mechanism. These differences are due to the strong deacylating effect of specific substrate in its complex with the enzyme modified with nonspecific acyl residue. The latter fact is suggested to be an experimental confirmation of the "induced fit" hypothesis.

Amidohydrolases↗

PubMedDate not supplied

Sperm-bound amidase activity as a marker for acrosomal integrity in bull spermatozoa.

An assay for sperm-bound amidase activity was validated using bovine spermatozoa and N-benzoyl-DL-arginine p-nitroanilide as substrate. The assay had intra- and interassay coefficients of variations of 5 and 12%, respectively. It is an inexpensive, simple and rapid assay since 100 samples can be evaluated in 2 hours and it requires only 4 X 10(6) spermatozoa per sample. Sperm-bound amidase activity was proportional (r = 0.95) to the percentage of spermatozoa with an intact acrosome, as determined by differential interference-contrast microscopy. A change of five percentage units in the incidence of damaged spermatozoa was detectable. Using this procedure, assessment of sperm-bound amidase activity is therefore a sensitive and efficient means of evaluating acrosomal integrity.

Acrosome↗

PubMed1994

Effects of environmental factors and Mn, Zn, Cu trace elements on the soil phosphomonoesterase and amidase activity. Application of DISITOBI model.

A soil incubation experiment was carried out in a complex experimental system to simulate the effect of environmental factors and 3 trace elements on the phosphomonoesterase and amidase activity of 2 soils. DISITOBI model assures information about experimental object characterized by multidimensional response-function. Plant residue+mineral nitrogen addition increased phosphomonoesterase activity of investigated soils through its effect on enzyme synthesis. A significant part of the increase in phosphomonoesterase activity is the result of the exogenous enzyme input of plant residues. Mineral phosphorus addition reduced the activity after 16 days incubation under our experimental conditions. Plant residue+mineral nitrogen addition reduced amidase activity of chernozem soil presumably due to ammonium and nitrate inhibiting soil amidases.

Amidohydrolases↗

PubMed1993

Stereoselectivity in glutathione conjugation and amidase-catalyzed hydrolysis of the 2-bromoisovalerylurea enantiomers in the single-pass perfused rat liver.

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of [(R)- and (S)-]2-bromoisovalerylurea (BIU), yielding bromoisovaleric acid (BI) and urea, have been observed in the rat in vivo and in isolated rat hepatocytes. The metabolism of enantiomeric (R)- and (S)-BIU was presently examined in the single-pass perfused rat liver with varying input concentrations (8-250 microM). Steady-state hepatic extraction ratios for (R)-BIU (0.6) were constant and higher than those for (S)-BIU, whose extraction ratio decreased from 0.36 (8 microM) to 0.23 (236 microM). (R)- and (S)-BIU were excreted unchanged only minimally into bile. [14C-Urea](R)-BIU underwent amidase-catalyzed hydrolysis to yield [14C]urea (15-24% of rate in) and conjugation to form the (S)-glutathionyl conjugate (31-35% of rate in); two metabolites, most likely the cysteinyl and dipeptide conjugates of BIU (10% of rate in), were found. [3H-Isovaleryl](S)-BIU formed much less amidase-hydrolyzed product, [3H]BI (1-2% of rate in) less (R)-glutathionyl conjugate (9-18% of rate in), but appreciable amounts (14-17% of rate in) of three other metabolites, of which two were most likely the cysteinyl and glycinylcysteinyl conjugates of BIU. When the glutathione conjugation products (glutathione, cysteine and cysteinylglycine conjugates) were summed, the total glutathione conjugation rate for (R)-BIU (44% of rate in) exceeded that for (S)-BIU (34 to 24% of rate in). Fitting of data to the Michaelis-Menten equation revealed similar Km for glutathione conjugation, but a 2-fold higher Vmax for (R)-BIU.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗

PubMed2004

Combinatorial formulation of biocatalyst preparations for increased activity in organic solvents: salt activation of penicillin amidase.

A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.

1-Propanol↗

PubMed1979

Properties of penicillin amidase immobilized by copolymerization with acrylamide.

An enzyme preparation in a spherical granule form was obtained by copolymerization of penicillin amidase (EC 3.5.1.11) (previously modified with maleic anhydride) and acrylamide via a crosslinking agent. As compared with the native enzyme, immobilized amidase is more resistant to heating, has a lower affinity to benzylpenicillin, and is less inhibited by phenylacetate. Its substrate specificity and optimum pH remain unchanged.

Acrylamides↗

PubMed2002

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery.

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.

Amino Acid Motifs↗

PubMed1991

N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain.

A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.

Alanine↗

PubMed1981

Activity of N-acetylmuramoyl-L-alanine amidase in phospholipidic environments.

A purified preparation of N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), a murein hydrolase from Escherichia coli, was found to lose its activity during incubation in the presence of bacterial phospholipid suspensions. Whether it was co-dispersed with the phospholipids or added to sonicated phospholipid suspension, the enzyme was inhibited (or inactivated) from the first minutes of incubation at 37 degree C. As phosphatidylglycerol/cardiolipin ratio of the phospholipid suspension as increased (all other things being equal), a further decrease of amidase activity was observed. The highest losses of activity were found after co-dispersion of the enzyme and the substrate together with the phospholipids, the resulting suspension being formed of larger multilayered vesicles, as revealed by electron microscopy. In these conditions, the effect on enzyme activity was only partially accounted for by the proportion of the enzyme that was entrapped in the vesicles. The entrapment capacity of the enzyme (using a 35S-labelled enzyme preparation) and of the substrate (3H-labelled) by the multilamellar phospholipidic vesicles did not significantly change as a function of their relative content of phosphatidylglycerol and cardiolipin. The possible physiological meaning of the results is discussed is connection with our previous data and with other works related to membranous phospholipid distribution and to septum formation control in bacteria.

Amidohydrolases↗