The cellular location of nitrilase and amidase enzymes of Brevibacterium R312

Abstract Growth of Brevibacterium R312 on acetonitrile results in the appearance of acetate and ammonia in the medium, whereas incubation of harvested bacteria with acetonitrile results in appearance of acetamide, acetate and ammonia in the medium. Acetonitrilase and acetamidase activities were found to be located in the cytosolic fraction of the cell, suggesting that acetonitrile and acetamide readily enter and leave the bacterium. This was confirmed by the inability of these compounds to osmotically stabilise protoplasts, whereas acetate does so. The small size and neutral non-ionic structure of acetonitrile and acetamide suggest they probably permeate by simple diffusion.     

Induction and Repression of Amidase Enzymes in Aspergillus nidulans

Aspergillus nidulans can grow on acetamide as both a carbon and nitrogen source and can also grow on formamide as a nitrogen source. Two distinct enzymes, an acetamidase and a formamidase, are produced. The control of the synthesis of these two enzymes in a wild-type strain was investigated. The formamidase is induced by acetamide and formamide and repressed by ammonia. The acetamidase is induced by formamide and acetamide, repressed by carbon metabolites derived from glucose and acetate, and repressed by ammonia. Repression of the acetamidase by ammonia depends on the carbon source; growth on glucose but not on acetate or acetamide allows repression to occur. The pattern of acetamidase repression is compared with that of histidine catabolic enzymes in various bacteria.     

Methane production from acetamide in an upflow anaerobic sludge-blanket reactor based on a synergistic association between an aerobic rod and methanogens

Acetamide degradation was investigated in a bench-scale upflow anaerobic sludge-blanket (UASB) reactor, successively fed with acetamide, acetate and acetamide, over a period of 343 days, at different hydraulic retention times (t _HR). The reactor was seeded with the sludge previously described [Guyot et al. (1994) Appl Microbiol Biotechnol, 42:452-456], in which methanogenesis from acetamide was performed through a synergistic relationship between an acetamide-degrading, aerobic rod and methanogens. When the reactor was fed acetamide, the chemical oxygen demand (COD) removal efficiency was 86% at volumetric loads less than 1.18 kg COD m^–3 day ^–1. At higher volumetric loads, the efficiency decreased markedly, e.g. 50.9% at a volumetric organic load of 3.39 kg COD m^–3 day^–1 (1 day t _HR) with an accumulation of both acetamide and acetate. The same reactor, when fed with acetate at t _HR 1 day, reached a high COD removal (99%). Evidence of the inhibition of acetate degradation by acetamide is presented. After a long period (135 days) without feeding the reactor with acetamide, the sludge reactor was still capable of degrading acetamide when this substrate was supplied again. It seems that the synergistic degradation of acetamide by aerobes and methanogens present in the UASB reactor sludge is stable over a long period (343 days), in spite of limiting concentrations of dissolved oxygen in the feed.     

Induction of the acetamidase of Aspergillus nidulans by acetate metabolism.

Growth tests and enzyme determinations strongly suggest that the acetamidase of Aspergillus nidulans is induced by a product of acetate metabolism rather than the substrate, acetamide. The cis-dominant mutation, amdI9, which is closely linked to amdS, the structural gene for the acetamidase, results in greatly increased sensitivity to induction by acetate metabolism. Propionate, L-threonine, and ethanol also result in acetamidase induction. Mutations in the facA, facB, and facC genes, which lead to low levels of acetyl-coenzyme A synthase, are epistatic to the amdI9 mutation for strong growth on acetamide medium and abolish acetamide and propionamide induction of the acetamidase and isocitrate lyase enzymes. Acetate, L-threonine, and ethanol, however, can induce these enzymes in strains containing facA and facC lesions but not in strains containing a facB lesion. The evidence suggests that acetamidase and isocitrate lyase may be induced by a similar mechanism.     

A sex difference in hepatic glutathione S-transferase B and the effect of hypophysectomy.

[2-14C]Methyl cyanide (acetonitrile) is metabolized to citrate, succinate, fumarate, malate, glutamate, pyrrolidonecarboxylic acid and aspartate. Non-radioactive acetamide and acetate compete with 14C from methyl cyanide, and [2-14C]acetate and [2-14C]methyl cyanide are metabolized at similar rates, giving identical products. This evidence, combined with the inhibitory effect of fluoroacetate and arsenite on methyl cyanide metabolism, indicates that the pathway is: methyl cyanide leads to acetamide leads to acetate leads to tricarboxylic acid-cycle intermediates. The pathway was investigated in a species of Pseudomonas (group III; N.C.I.B. 10477), but comparison of labelling patterns suggests that it also exists in several higher plants.     

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.     

Amide metabolism of Chlamydomonas reinhardi

Chlamydomonas reinhardi can utilise the lower aliphatic amides (C₁–C₄) as nitrogen sources. Of these only acetamide can serve as a sole carbon source. The acetamide analogue F-acetamide kills cells after conversion to F-acetate and F-citrate. This conversion is controlled by exogenous ammonia and, in part, acetate levels. Only one enzyme and one active site are involved in acetamidase function. Enzymatic analysis indicates an increased substrate range as compared to the growth — supported range, indicating uptake, toxicity or metabolic control restrictions.Abbreviations TCA trichloroacetic acid - TAP tris-acetate-phosphate medium - MIC mimmum inhibitory concentration - BSA bovine serum albumin     

Mycobacterium fortuitum subspecies acetamidolyticum, a new subspecies of Mycobacterium fortuitum

Mycobacterium fortuitum subspecies acetamidolyticum is a new subspecies of M. fortuitum and has an intermediate growth rate. It is a nonphotochromogenic mycobacterium. It does not utilize glutamate but utilizes acetamide as a simultaneous nitrogen and carbon source. It is able to utilize acetate, malate, pyruvate, fumarate, glucose, fructose, and n-propanol as the sole sources of carbon in the presence of ammoniacal nitrogen, but does not utilize them in the presence of glutamate-nitrogen. It is easily differentiated from all rapidly growing mycobacteria by its inability to utilize glutamate as a simultaneous nitrogen and carbon source, and from all slowly growing mycobacteria by its capacity to utilize acetamide as a simultaneous nitrogen and carbon source. Its mycolic acid pattern is different from that of M. fortuitum. However, its deoxyribonucleic acid showed 94% relatedness with that of M. fortuitum. In view of the above findings, it has been designated as a new subspecies of M. fortuitum. The organism was isolated from sputum of a 56-year-old patient with lung disease and is considered to be a lung pathogen. The type strain is ATCC 35931 (NCH E11620).     

Regulatory circuits of theamdS gene ofAspergillus nidulans

TheamdS gene codes for an acetamidase enzyme that hydrolyses acetamide to acetate and ammonium thus providingA. nidulans with a source of carbon and nitrogen. The exceptionally favourable genetics of this system combined with molecular analysis have enabled many regulatory circuits affectingamdS to be identified genetically. Characterization of the regulatory genes and the definition of the cis-acting sites involved have been done using bothin vivo andin vitro mutagenesis. Recent results on the analysis of the system are presented.     

Analysis of the Reaction between Formaldehyde and Amide

The reaction between formaldehyde and acetamide which affords a model compound for an amino acid having an amide group was analyzed to investigate the role of formaldehyde as a cross-linking reagent. One of the products was isolated by Sephadex G-10 column chromatography and was identified as N-hydroxymethyl acetamide (FA) by NMR spectrometry and mass spectrometry. Another product, which could not be isolated, was estimated to be N, N-dihydroxymethyl acetamide (F₂A) by kinetic analysis and mass spectrometry. The formation of N, N′-methylene diacetamide was not observed. The mechanism of the reaction between formaldehyde and acetamide was estimated by the kinetic analyses of NMR data, and the rate constants were calculated from the data by the optimization method with a digital computer. On the other hand, formaldehyde cross-linked product was obtained in the reaction of formaldehyde with acetamide and alanine, Its decomposing reaction was analyzed with an NMR spectrometer to study the stability of the formaldehyde cross-linked product. The degradation was dominantly initiated with the release of acetamide.

Consequently, it was estimated that the C–N bond between formaldehyde and amide is so labile that amide-bound formaldehyde does not react further with amides or amines, and that the amide-formaldehyde-amine condensation product is unstable and easily decomposes by releasing the amide.     

Increased and decreased sensitivity to carbon catabolite repression of enzymes of acetate metabolism in mutants of Aspergillus nidulans.

Summary The creA204, creB15 and creC27 mutations have been shown to cause carbon catabolite derepression of acetyl CoA synthase and isocitrate lyase in Aspergillus nidulans. A recessive mutation, cre-34, which is linked to the creC gene, results in these enzymes being more sensitive than cre or wildtype strains to catabolite repression. The acetamidase levels of strains containing cre mutations have been investigated and provide support for the hypothesis that an acetate metabolite, rather than acetamide, induces this enzyme.     

Membrane proteins and urea and acetamide transport in the human erythrocyte.

Previous studies have shown that urea and acetamide traverse the erythrocyte membrane by way of facilitated diffusion. The nature of this selective pathway is unknown. The present studies investigate the effects of proteolytic enzymes and crosslinking agents on amide transport. Cleavage of the erythrocyte membrane surface by pronase or trypsin had no effect on urea and acetamide permeability or inhibition by phloretin. These findings suggest that the sialoglycopeptide segment of the sialoglycoproteins is not critical to urea and acetamide transport. In addition, extensive crosslinking of membrane proteins with glutaraldehyde had no effect on amide transport in the absence or presence of phloretin.     

Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

槲寄生中的化学成分及其抗肿瘤活性(Ⅰ)

从槲寄生75%乙醇提取物中分离得到4个化合物,应用波谱学方法鉴定它们的结构为尼克酰胺(1),乙酰胺(2),齐墩果酸(3),β-香树脂醇乙酸酯(4).其中,化合物1和2为首次从槲寄生属植物中分离得到.采用MIT法对化合物1～4进行体外抗肿瘤活性研究,结果显示化合物1、2和4对HO-8910人卵巢浆液性腺癌、SMMC7721人肝癌、T24人膀胱癌、HepG2人肝癌和SHG人神经胶质瘤细胞系没有细胞毒活性,化合物3则对这五种肿瘤细胞系有显著的抑制作用.     

Differential handling of urea and its analogues suggests carrier-mediated urea excretion in freshwater rainbow trout

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.     

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

The reactivity and function of thiol groups in trout actin

1. Considerable differences were found between the rates and degrees of modification of native trout actin with iodo[2-(14)C]acetate and iodo[1-(14)C]acetamide. 2. With iodoacetate, G- and F-actin were both labelled in the N-terminal peptide only. This modification had little effect on the ability of the actin to polymerize. 3. Iodoacetamide labelled three cysteine residues in both G- and F-actin. The modified cysteine residues were identified from the position of the corresponding tryptic peptides on peptide ;maps'. 4. The modification had little effect on the ability of G-actin to polymerize, to bind ATP or to bind Ca(2+), or on the ability of F-actin to depolymerize. 5. It is concluded that the three cysteine residues present on the ;surface' of the native trout actin molecule have no direct role in the polymerization processes, the binding of ATP, or the binding of Ca(2+).     

Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from Aphelandra tetragona was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified. Fusarium sambucinum detoxified BOA and HBOA to N-(2-hydroxyphenyl)malonamic acid. Plectosporium tabacinum, Gliocladium cibotii, and Chaetosphaeria sp. transformed HBOA to 2-hydroxy-N-(2-hydroxyphenyl)acetamide, N-(2-hydroxyphenyl)acetamide, N-(2-hydroxy-5-nitrophenyl)acetamide, N-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3H-phenoxazin-3-one, 2-acetylamino-3H-phenoxazin-3-one, and 2-(N-hydroxy)acetylamino-3H-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-N-(2-hydroxyphenyl)[(13)C(2)]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to o-aminophenol as a key intermediate.