Biomedical subjects

A L Demain

Publications and source records attributed to A L Demain.

At least 73 records · Page 4Linked to original sources

PubMed1993

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, the multienzyme integrating the four primary reactions in beta-lactam biosynthesis, as a model peptide synthetase.

ACV synthetase forms the tripeptide precursor of penicillins and cephalosporins from alpha-aminoadipate, cysteine, and valine. Catalytic sites for substrate carboxyl activation as adenylates, peptide bond formations, epimerization and release of the tripeptide-thioester are integrated in multifunctional enzymes of 405 to 425 kD. These have been characterized from several pro- and eukaryotic beta-lactam producers. Implications of these results for the thio-template mechanism of peptide formation are discussed, as well as the use of this multienzyme as a model system for enzymatic peptide synthesis.

Amino Acid Sequence↗

PubMed1993

Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology.

It is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and SS) together degrade crystalline cellulose. SL is a glycoprotein of 210,000 Da which enhances the binding to cellulose and the activity of SS, an endoglucanase of 83,000 Da. We have previously reported the cloning of a DNA fragment encoding the N-terminal end of the SL protein using antibodies raised against the native protein. A chromosomal walking approach using an EcoRI and a Bam HI-Sau3A gene library allowed us to isolate the C-terminal end of the gene. Sequencing of both fragments revealed the existence of a leader peptide as has been found in cellulases of the same organism. This leader sequence is followed by a stretch of 14 amino acids that is identical to the N-terminal amino acid sequence of the native secreted protein. The open reading frame (ORF) of this gene encodes a protein of 196,800 Da and is followed by a hairpin loop that could be involved in transcription termination. Within the open reading frame (ORF), we found nine internal repeated elements (IREs) of about 500 nucleotides each. Seven of these sequences displayed 98-100% homology and were located adjacent to each other within the structural gene without intervening regions. The remaining two, located on the N-terminal end of the gene, showed a significantly lower homology. Bearing in mind the inherent instability of reiterated regions, we confirmed the authenticity of our clones by Southern blot analysis using chromosomal C. thermocellum DNA and ruled out the possibility of rearrangements during the cloning and sequencing process. The sequenced gene is designated cipA and the encoded SL protein CipA.

Amino Acid Sequence↗

PubMed1993

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.

A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SalI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcI). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative beta-lactamase activity was detected in S. lividans.

Amino Acid Sequence↗

PubMed1992

Biochemical studies on the activity of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.

2-Aminoadipic Acid↗

PubMed1992

Purification and characterization of a new endoglucanase from Clostridium thermocellum.

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.

Amino Acid Sequence↗

PubMed1992

Microbial secondary metabolism: a new theoretical frontier for academia, a new opportunity for industry.

Microbial secondary metabolites are the low molecular mass products of secondary metabolism. They include antibiotics, pigments, toxins, effectors of ecological competition and symbiosis, pheromones, enzyme inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, antitumour agents and growth promoters of animals and plants. They have a major effect on the health, nutrition and economics of our society. They have unusual structures and their formation is regulated by nutrients, growth rate, feedback control, enzyme inactivation and induction. Regulation is influenced by unique low molecular mass compounds, transfer RNA, sigma factors and gene products formed during post-exponential development. The synthases of secondary metabolism are often coded by clustered genes on chromosomal DNA and infrequently on plasmid DNA. The pathways of secondary metabolism are still not understood to a great degree and thus provide a new frontier for basic investigations of enzymology, control and differentiation. Cloning and expression of genes in industrial microorganisms offer new opportunities for strain improvement and discovery. Microbial metabolites have already established themselves as coccidiostats, immunosuppressants, antihelminthic agents, herbicides and cholesterol-reducing drugs. Great potential exists for the discovery of antiviral, antiparasitic, antitumour and pharmacological compounds and new agricultural products. The future for natural products is bright indeed.

Bacteria↗

PubMed1992

Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system.

A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS). By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant. Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis. This enabled us to recover viable cells after sorting. After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting. Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain. This method has the advantage of being able to screen large numbers of cells in a short time. Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.

Bacillus↗

PubMed1992

ACV synthetase.

ACV synthetase (ACVS) is the first enzyme and plays a key role in the biosynthesis of all natural penicillins and cephalosporins. The enzyme is extremely unstable and little had been known about it until recently. This article summarizes the progress in research on this enzyme, including the establishment of a cell-free assay system, stabilization, purification, characterization, and gene cloning. A possible reaction sequence for ACVS catalysis is suggested.

Acremonium↗

PubMed1991

Purification and characterization of cephalosporin 7 alpha-hydroxylase from Streptomyces clavuligerus.

Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.

Cephalosporins↗

PubMed1991

Purification and characterization of endoglucanase Ss from Clostridium thermocellum.

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

Blotting, Western↗

PubMed1991

Inhibition of growth of Bacillus subtilis by recombinant plasmid pCED3.

The present study deals with the mechanism by which plasmid pCED3 interferes with the growth of Bacillus subtilis. Plasmid pCED3 was constructed from pUB110 and pBR322 and contains the lacZ gene attached to the B. subtilis tms promoter. Plasmid derivatives that contain mutations in the tms promoter were used to examine the effect of promoter strength on cell growth, plasmid stability, the amount of plasmid DNA per cell and the activities of plasmid-encoded enzymes, i.e., beta-galactosidase and kanamycin nucleotidyltransferase (KNT). Efficient lacZ transcription directed from the tms promoter resulted in reduction in growth rate and plasmid stability without an increase in beta-galactosidase activity. The amount of plasmid DNA varied between 6.6 and 12.9 pmol per mg cell protein and showed no clear correlation with the strength of the tms promoter. Transcription from the tms promoter inhibited the expression of the plasmid-encoded kanamycin resistance gene resulting in the reduction of both beta-galactosidase activity and growth rate in the presence of kanamycin. These results suggest that the negative effect on B. subtilis growth exerted by pCED3 results at least partly from a decrease in kanamycin resistance by plasmid-bearing cells.

Bacillus subtilis↗

PubMed1991

Cloning and expression of a Clostridium thermocellum DNA fragment that encodes a protein related to cellulosome component SL.

Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C. thermocellum chromosomal DNA fragments constructed in the vector lambda gt11. A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated. No similarity with other previously cloned DNA fragments has been found. The anti-SL crossreacting polypeptide was isolated from recombinant Escherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity. Using CMC as the substrate, a temperature optimum of 55 degrees C and a pH optimum of 6.6 were observed. These properties were compared to those of C. thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities. In addition, the SL proteins produced in C. thermocellum and E. coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.

Cellulase↗

PubMed1991

Regulation of ACV synthetase in penicillin- and cephalosporin-producing microorganisms.

ACV synthetase is the first enzyme in the biosynthetic pathway for all natural penicillins and cephalosporins. Its activity catalyzes the possible rate-limiting step and is subject to various regulatory controls. In both the fungus Cephalosporium acremonium and the actinomycete Streptomyces clavuligerus, formation of the enzyme is repressed by ammonium and phosphate ions, but not by easily-utilized carbon sources; it is induced by methionine in C. acremonium. The action of the crude enzyme is indirectly inhibited in vitro by sugars such as glucose and by the carbon source metabolite glyceraldehyde-3-phosphate (G3P). Sugars are not inhibitory to the purified enzyme activity but G3P is inhibitory. The sugar inhibition is reversed by ATP and the G3P inhibition by L-cysteine (L-cys). Addition of L-cys to fermentation media increases beta-lactam production by both microorganisms. Phosphate and ferrous ions inhibit enzyme activity. Dissolved oxygen levels do not affect enzyme formation. Regulation of ACVS formation most likely occurs at the transcriptional level.

Journal Article↗

PubMed1991

Production of beta-lactam antibiotics and its regulation.

The discovery of penicillin was announced over 60 years ago. It was the first beta-lactam antibiotic and the importance of this group is greater today than it has ever been. It is clear that even at 60 years of age, beta-lactams are going strong and no one contemplates their early retirement. Currently, sales of beta-lactam compounds form the largest share by far of the world's antibiotic market. The beta-lactam antibiotics include penicillins such as penicillin G, penicillin V, ampicillin, cloxacillin, and piperacillin; cephalosporins such as cephalothin, cephaloridine, cephalexin, and cefaclor; and cephamycins such as cefoxitin. In addition, beta-lactam antibiotics include the more recently developed nonclassical structures such as monobactams, including aztreonam; clavulanic acid, which is a component of the combination drug augmentin; and thienamycin, which is chemically transformed into imipenem, a component of the combination drug known as primaxin (or tienam). The classical beta-lactam antibiotics can be divided into hydrophobic and hydrophilic fermentation products. The hydrophobic members, e.g. benzylpenicillin (penicillin G) and phenoxymethylpenicillin (penicillin V), contain non-polar side chains, e.g. phenylacetate and phenoxyacetate, respectively, and are made only by filamentous fungi; the best known of these is Penicillium chrysogenum. The antibacterial spectrum of the hydrophobic penicillins is essentially Gram-positive. The hydrophilic types are penicillin N, cephalosporins and 7-alpha-methoxycephalosporins (cephamycins) which are made by fungi, actinomycetes and unicellular bacteria. They all contain the polar side chain, D-alpha-aminoadipate. We can draw a sequence of reactions which describes the biosynthesis of all penicillins and cephalosporins, however the total sequence exists in no one microorganism. All penicillin and cephalosporin biosynthetic pathways possess the first three steps in common and all cephalosporin pathways go through deacetylcephalosporin C. However, there are many subsequent biosynthetic reactions which vary in the different producing organisms. Production of beta-lactam antibiotics occurs best under conditions of nutrient imbalance and at low growth rates. Nutrient imbalance can be brought about by limitation of the carbon, nitrogen or phosphorus source. In addition to these factors, amino acids such as lysine and methionine exert marked effects on production of penicillins and/or cephalosporins by some microorganisms. Induction of some of the synthetases, especially the first enzyme, ACV synthetase, by methionine is the basis of the methionine stimulation of cephalosporin C synthesis in C. acremonium. Inhibition of homocitrate synthase is the mechanism involved in lysine inhibition of penicillin synthesis in Penicillium chrysogenum.(ABSTRACT TRUNCATED AT 400 WORDS)

Acremonium↗

PubMed1990

Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins.

The stability of the unstable enzyme, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Cephalosporium acremonium C-10, was increased 10-fold which facilitated its purification. The active enzyme was purified over 100 fold to electrophoretic homogeneity by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography. It appears to have a minimal size of 360 kDa based on SDS-polyacrylamide gel electrophoresis.

Acremonium↗

PubMed1990

Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585.

We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various beta-lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicillin N synthase gene of the cephalosphorin pathway.

Bacterial Proteins↗

PubMed1990

Regulation of biosynthesis of bacilysin by Bacillus subtilis.

Production of the dipeptide antibiotic bacilysin by Bacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6 x 10(-4) M.

Amino Acids↗

PubMed1990

Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis.

Recombinant plasmid pCED3 [confers beta-galactosidase production (LacZ+) and kanamycin resistance (Kmr)] in Bacillus subtilis was found to be both segregationally and structurally unstable. Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media. Culture instability was found to be highest in complex and defined media supporting high growth rates. Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate. Slowing down growth by decreasing temperature was much less effective. A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.

Bacillus subtilis↗