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[structure: see text] The synthesis of a simplified thioglycosidic analogue (2) of mycothiol (1) is described. Evaluation of 2 against mycothiol S-conjugate amidase from Mycobacterium tuberculosis reveals good specific activity (7500 nmol min(-)(1) mg-protein(-)(1), vs 14 200 for 1), indicating that 2 can serve as a starting point for antitubercular drug design.
Fluoroacetate administered intraperitoneally (ip) to rats and mice is defluorinated to give fluoride ion evident in urine and kidney by 19F NMR. The use of [2-13C]-, [1,2-13C]-, and [1,2-14C]fluoroacetate, prepared from isotopically labeled glycine, combined with 13C NMR and TLC radioautography, respectively, reveals a complex mixture of urinary metabolites including an S-(carboxymethyl) conjugate complex in rats and mice and sulfoxidation products thereof in rats. Direct 13C NMR examination of the bile following treatment with [2-13C]fluoroacetate shows the presence of S-(carboxymethyl)glutathione or a related conjugate and an O-conjugate of fluoroacetate. Incubation of [13C]fluoroacetate with rat and mouse liver cytosol involves formation of S-[( 13C]carboxymethyl)glutathione and fluoride ion. Fluorocitrate is also detected by 19F NMR examination of fluoroacetate incubations with mouse liver cytosol. Fluoroacetamide administered ip to rats and mice yields urinary fluoride ion formed via fluoroacetate which is liberated on hydrolysis by an organophosphate-sensitive amidase. 19F NMR chemical shifts of other metabolites of fluoroacetamide are consistent with fluoroacetohydroxamic acid in the liver of mice and fluorocitrate in the urine of rats. Fluoroethanol gives urinary fluoroacetate and fluoride ion in rats and mice and is converted to fluoroacetaldehyde by mouse and rat liver microsomes. (-)- and (+)-erythro-fluorocitrates administered ip to rats yield mostly the parent compounds in urine at 6 h with increasing amounts of fluoride ion thereafter. 19F NMR establishes that rat and mouse liver cytosols defluorinate (-)- but not (+)-erythro-fluorocitrate and pig heart aconitase also defluorinates (-)-erythro-fluorocitrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present. The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems. Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.
Dormant, bacterial endospores are the most resistant living structures known. The spore cell wall (cortex) maintains dormancy, core dehydration, and heat resistance. The cortex peptidoglycan has a unique, spore specific structure that enables it to fulfill its role. The cross-linking index of spore cortex peptidoglycan is very low, occurring at only 2.9% of the muramic acid residues compared to 33% in vegetative cells. The level of cross-linking of the cortex may be important in maintaining spore dormancy and heat resistance. Approximately 50% of the muramic acid residues in spore cortex are substituted with muramic delta-lactam. This modification is spore specific and is the major characteristic feature of the cortex. The muramic delta-lactam has no apparent role in establishing core dehydration, maintaining dormancy or heat resistance. However, the muramic delta-lactam residues are necessary for spore cortex hydrolysis during germination. They constitute part of the substrate recognition profile of the germination specific lytic enzymes (GSLEs) which are responsible for cortex hydrolysis. Germination results in loss of dormant spore properties and hydrolysis of the cortex is essential for later germination events and outgrowth. Application of muropeptide analysis to determine peptidoglycan structural dynamics during germination has revealed an unexpected degree of complexity in peptidoglycan hydrolysis. At least three hydrolytic activities, an N-acetyl glucosaminidase, a lytic transglycosylase and a possible amidase, are involved. A non-hydrolytic activity, likely to be an epimerase of muramic acid also occurs early during germination. The lytic transglycosylase generates anhydro-muropeptides which are released during germination and may be recycled during outgrowth to form part of the new vegetative cell wall.
PURPOSE: Anandamides have been observed to lower intraocular pressure in the rabbit eye, preceded by a period of hypertension. Amidases are thought to catabolize these compounds into their component parts, including arachidonic acid. Direct application of arachidonic acid has been observed to cause a marked rise of intraocular pressure. Thus, anandamide analogs resistant to catabolism were thought possibly devoid of this initial hypertension, and their effects on rabbit IOP investigated. METHODS: A series of chiral alpha-substituted anandamides were synthesized and studied for their effect on the intraocular pressure (IOP) of normotensive pigmented rabbits. Each test compound was dissolved in an aqueous 2-hydroxypropyl-beta-cyclodextrin solution (containing 3% polyvinyl alcohol) and administered (62.5 micrograms) unilaterally to the eye. RESULTS: The most promising compounds caused a statistically significant reduction of IOP (vs. vehicle) in the treated eyes. Of these, the R-alpha-isopropyl compound exhibited the best activity tested. Unlike the alpha-unsubstituted analogs previously studied, hypotensive effects were not preceded by an initial elevation of IOP and indomethacin pre-treatment (12.5 mg, s.c.) did not eliminate the IOP response, as demonstrated by administered R-alpha-isopropyl anandamide. CONCLUSIONS: Catabolism of alpha-unsubstituted anandamides may account for their observed intraocular hypertensive effects. The physiological mechanism by which alpha-substituted anandamides work apparently differs from that of the more easily metabolized alpha-unsubstituted compounds.
A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100-6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis, were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4 degrees C), salinities (0-4% NaCl) and pressures (40-60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomyces points to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase-amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolis strains recommends their further development as industrial biocatalysts.
Considerable evidence indicates that the connecting tubule cells, a type of cell of the distal nephron which seems to participate on potassium secretion, may be the place where renal kallikrein is synthetized. As potassium secretion and kallikrein synthesis may occur in the same cells, we studied the effect of high potassium diet on renal kallikrein production. The kallikrein containing cells from rats fed a normal and high potassium diet were evaluated using a combination of morphometric analysis, conventional electron microscopy, and ultrastructural immunocytochemistry. High potassium diet produced hypertrophy and hyperplasia of the kallikrein containing cells. Hyperplasia was sustained by an increased number of immunoreactive cells/mm2 (151 +/- 14 vs. 86.4 +/- 12, P less than 0.01), an increased number of binucleated immunoreactive cells/mm2 (11.90 +/- 2.1 vs. 3.77 +/- 0.17, P less than 0.005), and by the presence of mitosis. Cell hypertrophy was sustained by an increased cross-sectional area of immunoreactive cells (mu 2) (320.4 +/- 21 vs. 104.5 +/- 6.1, P less than 0.001), by an increased area of basal plasma membrane infoldings, by an hypertrophy of the components of the Golgi complex, hypertrophy of the components of the rough endoplasmic reticulum, and by a larger number of secretory-like vesicles containing kallikrein. The rats fed with high potassium diet had higher values on urinary kallikrein excretion-amidase activity (3.70 +/- 0.51 vs. 2.01 +/-0.37 units/day, P less than 0.02), higher values on potassium excretion (18.8 +/- 1.7 vs. 1.31 +/- 0.1 mmol/day, P less than 0.001), and higher urinary volume (51.5 +/- 5.3 vs. 12.2 +/- 1.6 ml/day, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.
The zinc-dependent UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) that constitutes the outermost monolayer of Gram-negative bacteria. As LpxC is crucial for the survival of Gram-negative organisms and has no sequence homology to known mammalian deacetylases or amidases, it is an excellent target for the design of new antibiotics. The solution structure of LpxC from Aquifex aeolicus in complex with a substrate-analog inhibitor, TU-514, reveals a novel alpha/beta fold, a unique zinc-binding motif and a hydrophobic passage that captures the acyl chain of the inhibitor. On the basis of biochemical and structural studies, we propose a catalytic mechanism for LpxC, suggest a model for substrate binding and provide evidence that mobility and dynamics in structural motifs close to the active site have key roles in the capture of the substrate.
The endogenous cannabinoid agonist, anandamide produced a modest contractile response in guinea-pig isolated bronchus compared with the vanilloid receptor agonist capsaicin. The contractile response to both anandamide and capsaicin was inhibited by the vanilloid receptor antagonist, capsazepine. Furthermore, the NK(2)-selective antagonist, SR48968 but not the NK(1)-selective antagonist, SR140333 inhibited contractile responses to anandamide. The contractile response to anandamide was abolished in tissues desensitized by capsaicin. However, anandamide failed to cross-desensitize the contractile response to capsaicin. The contractile response to anandamide was not significantly altered in the presence of the CB(1) receptor antagonist, SR141716A, nor the amidase inhibitor, phenylmethylsulphonyl fluoride (PMSF) but was significantly increased in the presence of the neutral endopeptidase inhibitor, thiorphan. The cannabinoid agonist, CP55,940 failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) response in guinea-pig airways. In contrast, the ORL(1) receptor agonist, nociceptin, significantly inhibited this response. The results demonstrate that anandamide induces a modest contractile response in guinea-pig isolated bronchus that is dependent upon the activation of vanilloid receptors on airway sensory nerves. However, cannabinoid receptors do not appear to play a role in this regard, nor in regulating the release of neuropeptides from airway sensory nerves under physiological conditions.
1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.
1. We have studied the effect of palmitoylethanolamide (PEA, 2.5 - 30 mg kg(-1), i.p.) on upper gastrointestinal transit in control mice and in mice with chronic intestinal inflammation induced by croton oil. 2. PEA significantly and dose-dependently decreased intestinal transit. The inhibitory effect of PEA (10 mg kg(-1)) was not modified by the cannabinoid CB(1) receptor antagonist SR141716A (0.3 mg kg(-1), i.p.), the cannabinoid CB(2) receptor antagonist SR144528 (1 mg kg(-1), i.p.), N(G)-nitro-L-arginine methyl ester (L-NAME, 25 mg kg(-1), i.p.), yohimbine (1 mg kg(-1), i.p.), naloxone (2 mg kg(-1), i.p.) or hexamethonium (1 mg kg(-1), i.p.). 3. PEA levels were significantly decreased in the small intestine of croton oil-treated mice. In these animals, PEA also inhibited motility and this effect was not counteracted by SR141716A (0.3 mg kg(-1)), or SR144528 (1 mg kg(-1)). 4. Pre-treatment of mice with the amidase inhibitor phenylmethyl sulphonil fluoride (PMSF, 30 mg kg(-1), i.p.) did not modify the inhibitory effect of PEA, either in control or in mice with inflammation. 5. It is concluded that PEA inhibits intestinal motility with a peripheral mechanism independent from cannabinoid receptor activation. The decreased levels of PEA in croton oil-treated might contribute, at least in part, to the exaggerated transit observed during chronic intestinal inflammation.
The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent.
Highly enantioselective hydrolysis of alpha,alpha-disubstituted malononitriles by the strain Rhodococcus sp. CGMCC 0497 expressing both nitrile hydratase and amidase activity to give (R)-alpha,alpha-disubstituted malonamic acids which could be converted to valuable (R)- or (S)-alpha-alkylated amino acids are reported and the yields of the products are improved remarkably at a lower reaction temperature.
PEGA supports functionalised with permanent charges show superior swelling properties in aqueous media when compared to neutral PEGA; a novel positively charged PEGA resin significantly improves penicillin G amidase (PGA) catalysed biotransformation on solid support, by favouring accessibility of the negatively charged enzyme.
Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purified to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln(195), Ser(199), Cys(207) and Lys(213) of the common signature sequences were selected for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the k(cat) value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys(213), in the signature sequences, also displayed significant reductions (approx. 380-fold) in k(cat) value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln(195), Ser(199) and Lys(213) are involved.
1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.
1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs.