ABSTRACT
Se presentan tres casos de pacientes anoftálmicos con implantes orbitarios expuestos. Aunque solo un paciente mostraba signos clínicos evidentes de infección, los tres implantes fueron estudiados para determinar la presencia de microorganismos adheridos a su superficie mediante microscopia electrónica de barrido (MEB) y cultivo microbiológico. La MEB permitió la visualización de microorganismos adheridos a los tres implantes, si bien solo uno de ellos presentó crecimiento en los cultivos microbiológicos. Además, la técnica de MEB empleada en el caso número 3 consiguió una mejor orientación y apreciación de los microorganismos respecto a las imágenes de los casos número 1 y 2. Estos hallazgos apoyan la idea de que la superficie de los implantes orbitarios expuestos está colonizada por microorganismos incluso cuando todavía no muestran signos evidentes de infección. Por lo tanto, es necesario una eliminación mecánica de la superficie expuesta del implante antes de recubrirla con injertos o colgajos
Three cases are presented of anophthalmic patients with exposed orbital implants. Although only one patient showed evident clinical signs of infection, all three implants were studied to determine the presence of microorganisms adhered to their surface using a scanning electron microscopy (SEM) and microbiological culture. The SEM allowed the visualisation of microorganisms adhered to the three implants, although only one of them showed growth in the microbiological cultures. In addition, the SEM technique used in case No. 3 achieved a better orientation and appreciation of the microorganisms with respect to the images of cases No. 1 and 2. These findings support the idea that the surface of exposed orbital implants is colonised by microorganisms, even when they still do not show obvious signs of infection. Therefore, mechanical removal of the exposed surface of the implant is necessary before covering it with grafts or flaps
Subject(s)
Humans , Bacterial Adhesion , Orbital Implants/microbiology , Bacteriological Techniques , Micrococcus/ultrastructure , Microscopy, Electron, Scanning , Streptococcus pneumoniae/isolation & purificationABSTRACT
Three cases are presented of anophthalmic patients with exposed orbital implants. Although only one patient showed evident clinical signs of infection, all three implants were studied to determine the presence of microorganisms adhered to their surface using a scanning electron microscopy (SEM) and microbiological culture. The SEM allowed the visualisation of microorganisms adhered to the three implants, although only one of them showed growth in the microbiological cultures. In addition, the SEM technique used in case No.3 achieved a better orientation and appreciation of the microorganisms with respect to the images of cases No.1 and2. These findings support the idea that the surface of exposed orbital implants is colonised by microorganisms, even when they still do not show obvious signs of infection. Therefore, mechanical removal of the exposed surface of the implant is necessary before covering it with grafts or flaps.
Subject(s)
Bacterial Adhesion , Orbital Implants/microbiology , Adult , Anophthalmos , Bacteriological Techniques , Humans , Male , Micrococcus/ultrastructure , Microscopy, Electron, Scanning , Middle Aged , Streptococcus pneumoniae/isolation & purificationABSTRACT
In this study, the effect that 5 fermented broths of lactic acid bacteria (LAB) strains have on the viability or proliferation and adhesion of 7 potentially pathogenic microorganisms was tested. The fermented broth from Lactococcus lactis C660 had a growth inhibitory effect on Escherichia coli K92 that reached of 31%, 19% to Pseudomonas fluorescens, and 76% to Staphylococcus epidermidis. The growth of Staph. epidermidis was negatively affected to 90% by Lc. lactis 11454 broth, whereas the growth of P. fluorescens (25%) and both species of Staphylococcus (35% to Staphylococcus aureus and 76% to Staph. epidermidis) were inhibited when they were incubated in the presence of Lactobacillus casei 393 broth. Finally, the fermented broth of Lactobacillus rhamnosus showed an inhibitory effect on growth of E. coli K92, Listeria innocua, and Staph. epidermidis reached values of 12, 28, and 76%, respectively. Staphylococcus epidermidis was the most affected strain because the effect was detected from the early stages of growth and it was completely abolished. The results of bacterial adhesion revealed that broths from Lc. lactis strains, Lactobacillus paracasei, and Lb. rhamnosus caused a loss of E. coli K92 adhesion. Bacillus cereus showed a decreased of adhesion in the presence of the broths of Lc. lactis strains and Lb. paracasei. Listeria innocua adhesion inhibition was observed in the presence of Lb. paracasei broth, and the greatest inhibitory effect was registered when this pathogenic bacterium was incubated in presence of Lc. lactis 11454 broth. With respect to the 2 Pseudomonas, we observed a slight adhesion inhibition showed by Lactobacillus rhamnosus broth against Pseudomonas putida. These results confirm that the effect caused by the different LAB assayed is also broth- and species-specific and reveal that the broth from LAB tested can be used as functional bioactive compounds to regulate the adhesion and biofilm synthesis and ultimately lead to preventing food and clinical contamination and colonization of E. coli K92, B. cereus, and Ls. innocua.
Subject(s)
Bacteria/drug effects , Bacterial Adhesion/drug effects , Culture Media, Conditioned/pharmacology , Lactobacillaceae/metabolism , Microbial Viability/drug effects , Animals , Biofilms/drug effects , Escherichia coli/drug effects , Escherichia coli/growth & development , Lacticaseibacillus casei/metabolism , Lactococcus lactis/metabolism , Listeria/drug effects , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & developmentABSTRACT
In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 µg ml(-1). Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50-1,000 µg ml(-1)), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 µg ml(-1)). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Plant Extracts/pharmacology , Staphylococcus/drug effects , Biofilms/growth & development , Genistein/pharmacology , Hydroxybenzoates/pharmacology , Microbial Sensitivity Tests , Resveratrol , Skin Diseases, Bacterial/drug therapy , Staphylococcal Infections/drug therapy , Stilbenes/pharmacology , Vaccinium macrocarpon/chemistryABSTRACT
The N-acetyl-D-galactosamine (GalNAc) transport system of Escherichia coli K92 was studied when the bacterium was grown in a chemically defined medium containing GalNAc as a carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.0. Under these conditions, the uptake rate was linear for at least 3 min and the calculated Km for GalNAc was 3 microM. The transport system was strongly inhibited by sodium arsenate (70%), potassium cyanide (62%) and 2,4-dinitrophenol (75%). Analysis of bacterial GalNAc phosphotransferase activity revealed in vitro GalNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that GalNAc uptake depends on a specific phosphotransferase system. Study of activity regulation showed that N-acetylglucosamine and mannosamine specifically inhibit the transport of GalNAc in this bacterium. Analysis of expression revealed that the GalNAc transport system is specifically induced by GalNAc but not by N-acetylglucosamine (GlcNAc) or N-acetylmannosamine (ManNAc), two intimately related sugars. Moreover, full induction of GalNAc transport required the presence of both cAMP and GalNAc. Comparative studies revealed that E. coli K92 has developed a regulation mechanism that specifically induces the appropriate permease based on the presence of each respective phospho-amino sugar (GlcNAc, ManNAc and GalNAc). In this regulation system, GlcNAc is the preferred amino sugar as the carbon source. Finally, when E. coli K92 was grown using GalNAc, capsular polysialic acid production was strongly affected. The presence of intracellular phosphoderivative acetylamino sugars, generated by the action of the phosphotransferase transport system, can be responsible for this effect.
Subject(s)
Acetylgalactosamine/metabolism , Amino Sugars/metabolism , Escherichia coli/metabolism , Sialic Acids/biosynthesis , Acetylglucosamine/pharmacology , Culture Media , Cyclic AMP/pharmacology , Escherichia coli/drug effects , Hexosamines/pharmacology , Kinetics , Monosaccharide Transport Proteins/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolismABSTRACT
The N-acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37 degrees C in 50 mM Tris-HCl buffer, pH 8.0, containing 50 microg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K(m) for NeuAc was 0.1 microM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N'-dicyclohexyl-carbodiimide (50%), and 2,4-dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.
Subject(s)
Mannheimia haemolytica/metabolism , N-Acetylneuraminic Acid/pharmacokinetics , 2,4-Dinitrophenol/pharmacology , Animals , Arsenites/pharmacology , Biological Transport , Cations , Cattle , Coloring Agents/pharmacology , Dicyclohexylcarbodiimide/pharmacology , Dose-Response Relationship, Drug , Galactose/metabolism , Glucose/metabolism , Hydrogen-Ion Concentration , Kinetics , Serum Albumin/metabolism , Sodium Compounds/pharmacology , Temperature , Time Factors , Uncoupling Agents/pharmacologyABSTRACT
A new data treatment is described for designing kinetic experiments and analysing kinetic results for multi-substrate enzymes. Normalized velocities are plotted against normalized substrate concentrations. Data are grouped into n + 1 families across the range of substrate or product tested, n being the number of substrates plus products assayed. It has the following advantages over traditional methods: (1) it reduces to less than a half the amount of data necessary for a proper description of the system; (2) it introduces a self-consistency checking parameter that ensures the 'scientific reliability' of the mathematical output; (3) it eliminates the need for a prior knowledge of Vmax; (4) the normalization of data allows the use of robust and fuzzy methods suitable for managing really 'noisy' data; (5) it is appropriate for analysing complex systems, as the complete general equation is used, and the actual influence of effectors can be typified; (6) it is amenable to being implemented as a software that incorporates testing and electing among rival kinetic models.
Subject(s)
Enzymes/metabolism , Kinetics , Models, Chemical , Models, StatisticalABSTRACT
Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43 kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153 m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77 mM for CTP and 1.82 mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.
Subject(s)
Mannheimia haemolytica/enzymology , N-Acylneuraminate Cytidylyltransferase/metabolism , Amino Acid Sequence , Animals , Cattle , Chromatography, Affinity , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Female , Indicators and Reagents , Kinetics , Liver/enzymology , Mice , Molecular Sequence Data , Molecular Weight , N-Acylneuraminate Cytidylyltransferase/chemistry , N-Acylneuraminate Cytidylyltransferase/isolation & purification , Ovary/enzymology , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts.
Subject(s)
Amino Sugars/metabolism , Carbohydrate Epimerases/analysis , Carrier Proteins , Escherichia coli Proteins , Animals , Bacteria , Carbohydrate Epimerases/chemistry , Kidney/enzymology , Liver/enzymology , Oxo-Acid-Lyases/chemistry , Rats , Scintillation Counting , Spectrophotometry/methods , Swine , ThiobarbituratesABSTRACT
The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.
Subject(s)
Bacterial Capsules/metabolism , Mannheimia haemolytica/growth & development , Mannheimia haemolytica/metabolism , Sialic Acids/biosynthesis , N-Acylneuraminate Cytidylyltransferase/metabolism , Oxo-Acid-Lyases/metabolism , Sialyltransferases/metabolism , TemperatureABSTRACT
The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of the pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defined medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in the quantities of benzylpenicillin accumulated in the broths (between 1.8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.
Subject(s)
Coenzyme A Ligases/genetics , DNA, Bacterial/chemistry , Gene Expression Regulation, Enzymologic , Penicillin G/metabolism , Penicillium chrysogenum/metabolism , Pseudomonas putida/enzymology , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Models, Chemical , Molecular Sequence Data , Polymerase Chain Reaction , Pseudomonas putida/geneticsABSTRACT
The effect of phenylacetic acid (PAA) and several analogs on the activity of isopenicillin N synthase (IPNS) and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 has been tested. Whereas the substitution on the ring of a hydrogen atom by hydroxy-, methyl- or methoxy- groups did not cause any effect, the presence of halogens (Cl or Br) at positions 3 and/or 4 of PAA strongly inhibited these two enzymes. The replacement of hydrogen atoms by fluorine in certain positions also caused inhibition, but to a lesser extent.
Subject(s)
Acyltransferases/metabolism , Enzyme Inhibitors/pharmacology , Oxidoreductases/metabolism , Penicillin-Binding Proteins , Phenylacetates/pharmacology , Acyltransferases/antagonists & inhibitors , Bromine , Chlorine , Enzyme Inhibitors/chemistry , Fluorine , Oxidoreductases/antagonists & inhibitors , Phenylacetates/chemistryABSTRACT
Phenylacetic acid (PhAcOH) and 4-hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4-hydroxyphenylacetic acid-3-hydroxylase; and those blocked in the gene encoding homoprotocatechuate-2,3-dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc-CoA ligase protein; and those lacking this enzyme activity.
Subject(s)
Dioxygenases , Phenylacetates/metabolism , Pseudomonas putida/metabolism , Chromatography, High Pressure Liquid , Culture Media , DNA Transposable Elements , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mutagenesis , Mutation , Oxygenases/genetics , Oxygenases/metabolism , Pseudomonas putida/genetics , Pseudomonas putida/growth & developmentABSTRACT
Acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) from some plectomycete fungi is possibly involved in an accessory step of penicillin biosynthesis, in addition to its role in primary metabolism. We present the characterisation of the gene encoding this enzyme in Penicillium chrysogenum, which we designated acuA. Sequencing of genomic and cDNA clones showed that the coding region was interrupted by five introns, located at the same positions as those present in the Aspergillus nidulans homologue. This supports the possibility that the gene acquired its definitive mosaic organisation before the Penicillium/Aspergillus divergence. The mature transcript encodes a polypeptide with an M(r) of 74,287 which is 89.4% identical to its A. nidulans counterpart.
Subject(s)
Acetate-CoA Ligase/genetics , Ascomycota/genetics , Genes, Fungal , Introns , Penicillium chrysogenum/genetics , Amino Acid Sequence , Base Sequence , Conserved Sequence , DNA, Fungal , Molecular Sequence Data , Nucleic Acid Hybridization , Sequence Homology, Amino AcidABSTRACT
Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.
Subject(s)
Coenzyme A Ligases/isolation & purification , Pseudomonas putida/enzymology , Repressor Proteins , Saccharomyces cerevisiae Proteins , Caprylates , Coenzyme A Ligases/biosynthesis , Coenzyme A Ligases/chemistry , Coenzyme A Ligases/metabolism , Culture Media/chemistry , Enzyme Induction , Phenylacetates , Substrate SpecificityABSTRACT
Carbon catabolite repression and pH regulation are regulatory circuits with a wide domain of action in the Plectomycetes. Penicillin biosynthesis is one of the pathways which are under their control. The conclusions obtained so far, which are based on studies of the genetic and molecular regulation of the penicillin pathway of Aspergillus nidulans, would have been much harder to produce using an organism such as Penicillium chrysogenum (the industrial penicillin producer). However, A. nidulans and P. chrysogenum are close in terms of their phylogeny and one can reasonably predict that the conclusions about A. nidulans, which are summarized in this review and which are of unquestionable biotechnological relevance, will be extrapolable to the industrial organism.
ABSTRACT
Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.
Subject(s)
Acetate-CoA Ligase/isolation & purification , Acetate-CoA Ligase/metabolism , Penicillins/biosynthesis , Penicillium chrysogenum/enzymology , Aspergillus nidulans/enzymology , Chromatography, Affinity , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Kinetics , Molecular Weight , Penicillins/isolation & purification , ThermodynamicsABSTRACT
Forty-seven different penicillins, including some of great clinical importance, have been synthesized "in vitro" by coupling the newly described enzyme phenylacetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum. Incubations were carried out at 30 degrees C in 50 mM HCl-Tris buffer pH 8.0. The reaction mixtures contained 6-APA, CoA, ATP, dithiothreitol, Mg2+ and the corresponding penicillin side-chain precursor. This is the first description of the enzymatic synthesis of all the natural penicillins known, many of the semisynthetic until now reported, and some penicillins that could only be currently obtained by chemical synthesis. The efficiency of this prokaryotic-eukaryotic enzymatic-coupled system and its application to the synthesis of different beta-lactam antibiotics are discussed.
Subject(s)
Acyltransferases/metabolism , Coenzyme A Ligases/metabolism , Penicillin-Binding Proteins , Penicillins/biosynthesis , Acyltransferases/isolation & purification , Chemistry, Pharmaceutical/methods , Coenzyme A Ligases/isolation & purification , Penicillium chrysogenum/enzymology , Pseudomonas putida/enzymologyABSTRACT
Seven different penicillins containing alpha-ketobutyric, beta-ketobutyric, gamma-ketovaleric, alpha-ketohexanoic, delta-ketohexanoic, epsilon-ketoheptanoic, and alpha-ketooctanoic acids as side chains have been synthesized in vitro by incubating the enzymes phenylacetyl coenzyme A (CoA) ligase from Pseudomonas putida and acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum with CoA, ATP, Mg(2+), dithiothreitol, 6-aminopenicillanic acid, and the corresponding side chain precursor.
Subject(s)
Penicillins/biosynthesis , Keto Acids/chemistry , Microbial Sensitivity Tests , Micrococcus luteus/drug effects , Penicillins/chemistry , Penicillins/pharmacology , Pseudomonas putida/metabolism , Structure-Activity RelationshipABSTRACT
3-Furylmethylpenicillin was synthesized in vitro from 3-furylacetic acid, 6-aminopenicillanic acid (6-APA), CoA, ATP and Mg2+. The reaction was catalyzed in two steps by the enzymes phenyl-acetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum. PCL catalyzes the activation of 3-furylacetic acid to 3-furylacetyl-CoA (3-F-CoA) and AT acylates the amino group of 6-APA with the 3-furylacetyl moiety of 3-F-CoA, releasing CoA and 3-furylmethylpenicillin.