Aliphatic Amidase Research Articles

ETUDE DE L'ACETAMIDASE D'UNE SOUCHE DE BREVIBACTERIUM

The acetamidase of Brevibacterium R312, a strain with nitrilase activity, has very different characteristics from those of its acetonitrilase. This acetamidase presents an activity for pH values greater than 3 and has an optimum activity at pH 7. The optimal temperature is between 60° and 70°. The enzyme resists high temperatures and its Michaelis constant is 3.5×10-3M. The -SH, -OH, -NH2, -NH-C-NH2, and tryptophan NH groups seem to be necessary for the acetamidase activity. The enzyme remains in supernatant fluid from the centrifugation at 180, 000×g. It is an adaptative enzyme. This acetamidase looks like aliphatic amidases described by other authors.

Formamide: the minimum-structure substrate for urease

Formamide has been found to be a bona fide substrate for jackbean urease (urea amidohydrolase, EC 3.5.1.5) and can be hydrolyzed to completion by the enzyme. The pH maximum for the reaction was 5.2, the Michaelis constant was 0.5 (±0.05) M at pH 5.2 and at pH 6.8, and the maximal velocity was 50-fold lower than that for urea at pH 6.8 its pH maximum, which nevertheless represents the highest V for any of the second-substrates of urease. As a competitive inhibitor of urea at pH 6.8, formamide had a K i = 0.4 (±0.11) M, indicating that the Michaelis constants for both urea and formamide approximate the true dissociation constants for the respective enzyme-substrate complexes. The ratios of formamide/urea hydrolysis remained constant during purification of the enzyme to maximal specific activity; and formamide hydrolysis was completely inhibited in the presence of 0.8 mM acetohydroxamate, a specific urease inhibitor. Formamide did not produce a progressive inhibition of urease, as had been found to occur with two other substrates possessing the hydroxamate functional group: hydroxyurea and dihydroxyurea. The kinetic constants for urease hydrolysis of formamide compare favorably with those of a representative bacterial aliphatic amidase. Urease did not hydrolyze the closest homologs of formamide: N-methyl formamide and acetamide. fa ]∗|The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

Temperature‐Induced Metabolic Alterations in a Thermophilic Bacillus

The effect of growth temperature on activity and rate of synthesis of the α‐aminoisobutyric acid transport system and of several enzymes of Bacillus stearothermophilus var. non diastaticus was investigated. The uptake of α‐aminoisobutyrate, as well as the activities of alanine dehydrogenase and aliphatic amidase were found to be considerably reduced in cultures grown at the near maximal temperature for growth (65°C) as compared to those grown at the optimal temperature for growth (58°C), although the growth rates at both temperatures were similar. The reduced rate at 65°C was due to a decreased differential rate of synthesis of these activities, while the increase in the activities at 58°C was due to de novo synthesis of the enzymes and the α‐aminoisobutyrate transport system rather than to activation of pre‐existing inactive enzymes or permeases. The highest rate of synthesis of alanine dehydrogenase and of the α‐aminoisobutyrate‐uptake system was obtained in cells grown at the near minimal temperature for growth (42–45°C).The reduced rate of enzyme synthesis at the higher temperatures seemed to be due both to an increase in catabolite repression and to faulty oxidative metabolism during growth at these temperatures. The effects mentioned were only partly abolished in a mutant resistant to catabolite repression, when grown at the high temperatures. The thermoadaptation process is discussed in terms of energy production and ATP availability for protein biosynthesis.The alterations of the metabolic activities induced by the environmental temperature were reversible and represent a physiological thermoadaptative response of the cells.

Relationship between culture density and catabolite repression of an inducible aliphatic amidase in a thermophilic bacillus.

A direct correlation between the absorbance of a thermophilic bacillus and specific amidase activity was observed, which was found to depend on the cell density of the culture rather than on the time of contact of the culture with the inducer. Dilution of high density cultures caused the specific amidase activity to decrease. Environmental factors such as pH, concentration of inducer or degree of aeration, and level of NH+4 and glutamate had no effect on amidase synthesis. The decrease in amidase activity upon dilution could not be ascribed to destruction by oxygen or by inactivation or decay. Several lines of evidence suggest that catabolite repression is responsible for the phenomenon described. Succinate-grown cultures gave a stronger dilution effect thatn glutamate-grown cells. The mutant strain E-21, relatively resistant to catabolite repression, did not show the characteristic dilution effect nor the direct correlation between absorbance and specific amidase activity.

Regulatory properties of an inducible aliphatic amidase in a thermophilic bacillus.

A thermophilic bacillus growing on acetamide as both carbon and nitrogen sources produces an inducible amidase. This amidase hydrolysed the following amides in decreasing order or activity, in comparison with acetamide (1.00): propionamide (0.97), fluoroacetamide (0.84), formamide (0.35) and glycinamide (0.12). Cyanoacetamide, dimethylacetamide, dimethylformamide and urea also induced the synthesis of the amidase, but were not substrates of the enzyme. Studies with protoplasts suggest that the amidase is located in the cytoplasm. Glucose strongly inhibited amidase synthesis; and limiting nitrogen did not release this inhibition. Urea strongly inhibited amidase activity in a competitive manner; but the inhibition caused by iodoacetamide and cyanoacetamide was non-competitive. Both thioacetamide and thiourea were effective inhibitors of enzyme induction. Bacteria grown on a succinate-minimal medium exhibited a lag in amidase synthesis, which could be eliminated by decreasing the concentration of succinate. Acetate- or pyruvate-grown cultures behaved similarly, while those grown on alanine or glutamate exhibited no lag in enzyme induction. In the mutant strain E21, repression of amidase synthesis by glucose was much less evident and no lag for induction was apparent with any of the other carbon sources mentioned.

Catabolite Repression of Pseudomonas aeruginosa Amidase: The Effect of Carbon Source on Amidase Synthesis

Synthesis of the Pseudomonas aeruginosa aliphatic amidase was repressed severely by succinate and malate and less severely by glucose, acetate or lactate. Amidase synthesis in inducible and constitutive strains was stimulated by cyclic AMP, which also gave partial relief to catabolite repression produced by the addition of lactate to cultures growing in pyruvate medium. Mutants which were resistant to catabolite repression were isolated from succinate+lactamide medium.

Selective Inhibition and the Kinetic Mechanism of the Aliphatic Amidase of Pseudomonas aeruginosa

Selective Inhibition and the Kinetic Mechanism of the Aliphatic Amidase of <i>Pseudomonas aeruginosa</i>

Genetics and Enzymology of Chloramphenicol Resistance

P. cepacia strain 716. P.putida strain A90 was particularly interesting. It has proved impossible to detect any acetamidase activity in this strain and no acetamide-utilizing mutants have ever been isolated. However, it grows very well on phenylacetamide and thus resembles the acetamide-negative mutant of strain A87 and some of the phenylacetamide mutants of P. aeruginosa PACI. The phenylacetamidase of strain A90 has been purified and still gives no cross reaction with the P. aeruginosa aliphatic amidase antiserum. Further studies on these amidases may indicate whether the genes for the naturally occurring acetamidases and phenylacetamidases have a common ancestry or are of independent origin.

Assay for the enzymic hydrolysis of penicillin and acylhydroxamates

A rapid and easily reproducible assay for penicillin acylase and aliphatic amidase activity is described. The method, which is based on the colorimetric estimation of proton release, is very sensitive and rates as low as 0.5 nmoles/minute can be measured readily. Determination of the kinetic constants of the enzymes gave results as good as or better than standard procedures.

Transition-state analogs of an aliphatic amidase

The use of transition-state analogs in the elucidation of enzyme mechanisms has assumed importance in recent years [ 1,2] . The idea that synthetic compounds resembling the proposed transition-state should be extremely effective inhibitors compared to ground-state analogs, and should bind more effectively even than the substrate, has been substantiated. Recent studies with papain [3] and elastase [4] showing that aldehydes with the appropriate peptide side chains are extremely effective inhibitors has led to the proposal that the free aldehyde reacts with an amino acid residue (cysteine-papain; setie-elastase) in the active centre to form a hemithibacetal or hemiacetal; this being the ‘true’ transition-state analog on the reaction pathway to acylenzyme formation. Aldehydes, however, exist in appreciable amounts in aqueous solution as hydrates formed rapidly by a general base catalysed reaction [S] . At the present time there is no way of determining whether the true inhibitor is the free aldehyde or its hydrate. This paper presents evidence that, at least for the aliphatic amidase from Pseudomonas aeruginosa, it is the hydrated aldehyde that is the true inhibitor. T%is conclusion is substantiated by the observation that acetaldehyde-ammonia is a potent inhibitor and might be considered to be a transition-state analog for amide hydrolysis.

Growth of pseudomonas species on phenylacetamide.

SUMMARY: Wild-type strains of Pseudomonas putida, P. cepacia and P. acidovorans grew in minimal salts media with acetamide or phenylacetamide as the carbon or nitrogen source. Wild-type strains of P. aeruginosa utilized acetamide but not phenylacetamide. Pseudomonas putida A90 and mutants isolated from P. putida A87 and from P. cepacia 716 grew on phenylacetamide but not on acetamide. These activities were due to two types of amidase: (i) aliphatic amidases, which hydrolyse acetamide, produced by P. aeruginosa and strains of P. putida, P. cepacia and P. acidovorans; and (ii) phenylacetamidases, which hydrolyse phenylacetamide, produced by some strains of P. putida, P. cepacia and P. acidovorans.

Biochemical and immunological comparison of aliphatic amidases produced by Pseudomonas species.

Summary: Strains of Pseudomonas aeruginosa, P. putida, P. acidovorans and P. cepacia were tested for growth on minimal salt media containing aliphatic amides. All strains of P. aeruginosa, P. acidovorans, P. cepacia and some strains of P. putida biotype A grew on acetamide; some strains of P. putida and P. acidovorans also grew on butyramide. The inducible aliphatic amidases had both hydrolase and transferase activities and were similar in substrate specificities. All species hydrolysed propionamide more rapidly than acetamide while acetamide was the best substrate for the transferase reaction. The rate of butyramide hydrolysis was 2 to 3% of the rate of acetamide hydrolysis for all species including those strains able to grow on butyramide. The amidases of P. putida a87 and P. acidovorans 14 were inducible by butyramide. Cell-free extracts of P. aeruginosa strains induced with acetamide cross-reacted completely with antiserum prepared against purified P. aeruginosa amidase A. Partial cross-reactions occurred with P. putida and P. acidovorans amidases and a very weak cross-reaction with P. cepacia amidase.

Biochemical and genetic studies with regulator mutants of the Pseudomonas aeruginosa 8602 amidase system.

Surmmary Mutants of Pseudomonas aeruginosa strain 8602 were isolated which were unable to produce an aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) and could not grow on acetamide as a carbon or nitrogen source. Amidase-constitutive mutants, producing amidase in the absence of inducing amides, were isolated by selection on succinate+formamide agar. Sixteen mutants were magno-constitutive non-inducible mutants producing amidase at about the same rate or greater than the fully induced wild-type strain. Amidase synthesis in one magno-constitutive mutant was repressed by the non-substrate inducer N-acetylacetamide, but the others were not affected in any way. Six mutants were semi-constitutive, producing amidase at 10–50% of the rate of the magno-constitutive mutants and were induced by N-acetylacetamide. Most of the constitutive mutants were very sensitive to catabolite repression by succinate in pyruvate medium, but succinate produced only partial repression of one magno-constitutive mutant and three semi-constitutive mutants; one semi-constitutive mutant was not repressed except in the presence of inducer. Six mutants isolated from succinate + formamide agar had altered inducer specificity and were induced to form amidase by formamide, which is a very poor inducer for the wild-type strain. The formamide-inducible mutants were also sensitive to catabolite repression by succinate although one mutant was only partially repressed. Phage F 116 was used to transduce the amidase structural and regulator genes. In crosses between constitutive mutants of Pseudomonas aeruginosa as donors and amidase-negative mutants as recipients, the two characters were co-transduced with frequencies of 80–100%. Similarly, in crosses between formamide-inducible and amidase-negative mutants these two characters were co-transduced with frequencies of 89–96%. The amidase structural and regulator genes are considered to be closely linked.

The aliphatic acylamide amidohydrolase of Mycobacterium smegmatis: its inducible nature and relation to acyl-transfer to hydroxylamine.

SUMMARY Mycobacterium smegmatis nctc 8159 grew well in a minimal medium with succinate or acetamide as sole carbon source. Washed bacteria or cell-free extracts hydrolyzed 15 monocarboxylic amides, but not 4 related N-substituted amides. Formamide was the best substrate, followed by n-butyramide. Extracts of bacteria grown on acetamide hydrolyzed formamide about 60 times and butyramide about 20 times as rapidly as bacteria grown on succinate. Other short-chain fatty acylamides were also more rapidly hydrolyzed, but benzamidase activity was not similarly induced by growth on acetamide. Extracts of bacteria grown on succinate transferred acyl groups from propionamide, butyramide and nicotinamide to hydroxylamine, to form hydroxamates. Transferase activity, unlike aliphatic amidase activity in extracts was purified twofold and freed from transferase activity. Formamidase and butyramidase activities were not separated, and were similarly affected by heat and dithio-bis-nitrobenzoic acid. The amidase was induced by growth on acetate and on butyramide, but not on propionate, butyrate or benzamide, all of which were good growth-substrates. N-methylacetamide and N-acetyl-acetamide were non-substrate inducers of amidase for bacteria growing on succinate.