Biomedical subjects

J M Luengo

Publications and source records attributed to J M Luengo.

At least 55 records · Page 3Linked to original sources

PubMed1991

"In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system.

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.

Acyltransferases↗

PubMed1990

Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid.

A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.

Amino Acids↗

PubMed1990

Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida.

Phenylacetyl-CoA ligase (PA-CoA ligase) from P. putida U is a newly described enzyme involved in the aerobic catabolism of phenylacetic acid. The enzyme was specifically induced when P. putida was grown in a chemically defined medium containing phenylacetic acid as the sole carbon source. The induction of PA-CoA ligase was delayed by adding easily metabolizable carbon sources to the medium; the effect was more drastic in the presence of glucose. Glucose did not cause catabolic inactivation but rather catabolic repression, this effect being reversed by cAMP.

Aerobiosis↗

PubMed1990

Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzylpenicillin in vitro.

A hybrid (prokaryotic-eukaryotic) enzyme system leading to the production of benzylpenicillin has been developed. In vitro synthesis of penicillin G was achieved by incubating 6-aminopenicillanic acid, CoA, phenylacetic acid, homogeneously pure phenylacetyl-CoA ligase (PA-CoA ligase) from Pseudomonas putida and acyl-CoA:6-APA acyltransferase (AT) from Penicillium chrysogenum. Benzylpenicillin was also obtained when AT was coupled with PA-CoA ligase and isopenicillin N-synthetase (IPNS). This is the first description of an in vitro assay that, using enzymes of different microbial origin, mimics the three last enzymatic steps leading to the biosynthesis of penicillin G in P. chrysogenum.

Acyltransferases↗

PubMed1990

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature.

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.

Antigens, Bacterial↗

PubMed1990

Aliphatic molecules (C-6 to C-8) containing double or triple bonds as potential penicillin side-chain precursors.

Three different hexenoyl-CoA derivatives (trans-2-hexenoyl-CoA, trans-3-hexenoyl-CoA and trans-trans-2,4-hexadienoyl-CoA), two octenoyl-CoA (trans-2-octenoyl-CoA, trans-3-octenoyl-CoA) and 2-octynoyl-CoA were tested as substrates of the enzyme acyl-CoA: 6-Aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. Only trans-3-hexenoyl-CoA and trans-3-octenoyl-CoA were recognized by AT and efficiently converted into penicillin F and octenoylpenicillin, respectively. The Km values for these substrates were 0.6 and 0.5 mM, suggesting that the affinity of AT for these molecules is similar to that reported for phenyl acetyl-CoA, octanoyl-CoA and hexanoyl-CoA (0.5, 0.6, and 1 mM, respectively). The absence of enzymatic activity shown by AT with the other acyl-CoA derivatives tested is due to the different position of the double or triple bond(s) in their aliphatic chains. The influence of the free rotation round the bond C-2-C-3 and possibility of planar conformation in such molecules and the importance in the formation of the enzyme-substrate complex is discussed.

Acyl Coenzyme A↗

PubMed1990

Biosynthesis of benzylpenicillin (G), phenoxymethylpenicillin (V) and octanoylpenicillin (K) from glutathione S-derivatives.

"In vitro" synthesis of benzylpenicillin and phenoxymethylpenicillin has been carried out by direct N-acylation of 6-aminopenicillanic acid (6-APA) with S-phenylacetyl- and (S-phenoxyacetyl)glutathione. The reactions were catalyzed by the enzyme acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum and in both cases the synthesis of antibiotics was enhanced by CoA. Penicillin K, a natural penicillin, was also synthesized "in vitro" by incubating (S-octanoyl)glutathione, 6-APA and AT, but in this case the formation of antibiotic required the presence of CoA. Furthermore, benzylpenicillin was obtained from (S-phenylacetyl)cysteinylglycine and 6-APA, suggesting that some intermediates of the gamma-glutamyl cycle are directly involved in the biosynthesis of penicillins. To explain "in vivo" formation of this beta-lactam antibiotic, a biosynthetic pathway which includes several glutathione-S-derivatives and a non-enzymatic reaction, is proposed.

Acyltransferases↗

PubMed1989

Regulation of colominic acid biosynthesis by temperature: role of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37 degrees C. No activity was found in E. coli grown at 20 degrees C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20 degrees C were transferred to 37 degrees C and was halted when cells grown at 37 degrees C were transferred to 20 degrees C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.

Escherichia coli↗

PubMed1989

In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides.

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.

Cell Membrane↗

PubMed1989

Uptake of phenylacetic acid by Penicillium chrysogenum Wis 54-1255: a critical regulatory point in benzylpenicillin biosynthesis.

The transport system of phenylacetic acid (PA) in Penicillium chrysogenum was studied. Kinetic measurements were carried out "in vivo" at 25 degrees C in 0.06 M phosphate buffer at pH 6.5. Uptake was a linear function of time over 3 minutes and the Km was 5.2 microM. PA uptake was inhibited by 2,4-dinitrophenol, 4-nitrophenol, sodium azide, potassium cyanide. N-ethylmaleimide, amino acids, xylose and fatty acids whereas lactose and ribose stimulated it. Benzylpenicillin, phenoxymethylpenicillin, penicillins DF, K and 6-aminopenicillanic acid did not modify uptake whereas phenoxyacetic acid and many phenyl derivatives strongly inhibited the incorporation of PA. PA transport is an inducible system that is strictly regulated by the carbon source used for P. chrysogenum growth. Uptake is not induced by phenoxyacetic acid and is repressed by L-lysine. The absence of the PA transport system when P. chrysogenum is grown in the presence of readily metabolized sugars and its repression by L-lysine suggests that this is a critical regulatory point in the control of benzylpenicillin biosynthesis.

Amino Acids↗

PubMed1989

Phenylacetic acid transport system in Penicillium chrysogenum Wis 54-1255: molecular specificity of its induction.

The phenylacetic acid (PA) transport system of Penicillium chrysogenum is induced by PA, 2-hydroxyphenylacetic and 4-phenylbutyric acids but not by benzoic, phenoxyacetic acid and phenylpropionic acids. Substitution in the aromatic moiety (3-hydroxyphenylacetic, 4-hydroxyphenylacetic acids), replacement of the aromatic moiety by other rings (thiophene-2-acetic acid, indole-3-acetic or indole-3-butyric acids) or the presence of an amino group in the alpha-position (2-aminophenylacetic acid) eliminates inducing activity. 2-Phenylbutyric acid dose not induce the PA transport system indicating that fatty acid-beta-oxidation is needed to generate the authentic regulatory molecule (phenylacetyl-CoA) from 4-phenylbutyric acid. Furthermore, the uptake system synthesized in presence of PA, 2-hydroxyphenylacetic or 4-phenylbutyric acids is under carbon catabolic repression control and is also repressed by L-lysine suggesting that the three molecules induce in P. chrysogenum a single mechanism of transport.

Benzoates↗

PubMed1989

Repression of phenylacetic acid transport system in Penicillium chrysogenum Wis 54-1255 by free amino acids and ammonium salts.

The phenylacetic acid (PA) transport system in Penicillium chrysogenum is an inducible-system (see Fernández-Cañón et al.; preceding papers) which is repressed by free amino acids when these molecules are added to the complex fermentation broths at the induction time. L-Tyrosine, L-alpha-aminoadipic acid, L-tryptophan, L-phenylalanine and L-methionine are the molecules that cause the greatest delay in induction. The addition of Krebs-cycle intermediates to the complex fermentation broth did not affect the rate of induction with the exception of oxalacetic acid and citric acid which strongly increased it. Ammonium salts and acetate also repressed the biosynthesis of the enzymes involved in the PA uptake.

Amino Acids↗

PubMed1989

IV. Acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum: studies on substrate specificity using phenylacetyl-CoA variants.

Two different penicillins (p- and m-methylbenzylpenicillin) were obtained "in vitro" by direct enzymatic synthesis, using homogeneously pure acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase from Penicillium chrysogenum, 6-APA and p- or m-tolylacetyl-CoA. The Km for these substrates were 6 and 15 mM, respectively, indicating that the affinity of the enzyme for these two molecules is much lower that shown by phenylacetyl-CoA (0.55 mM). Furthermore, acyltransferase does not recognize o-tolylacetyl-CoA as a substrate suggesting that the position of the methyl group on the aromatic moiety may have a very important role in the formation of the enzyme-substrate complex.

Acetyl Coenzyme A↗

PubMed1988

A protein-sialyl polymer complex involved in colominic acid biosynthesis. Effect of tunicamycin.

A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.

Bacterial Proteins↗

PubMed1988

Enzymatic synthesis of penicillins.

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.

Acyltransferases↗

PubMed1987

Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system.

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.

Antimetabolites↗

PubMed1987

Separation by high-performance liquid chromatography of penicillins with C4 to C10 aliphatic side chains.

A suitable and rapid method for the simultaneous determination of different aliphatic penicillins is described. Butyryl-, pentanoyl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl- and decanoylpenicillin can be completely separated by high-performance liquid chromatography using an isocratic elution mode (50 mM H2KPO4, pH 5.0:methanol, 45:55 v/v). Under these conditions, retention times for C4 to C10 aliphatic side-chain penicillins were 2.5, 2.8, 4.1, 5.8, 8.9, 15.3, and 28.2 min. The benzylpenicillin retention time was 3.3 min.

Chromatography, High Pressure Liquid↗

PubMed1987

Interference by methionine on valine uptake in Acremonium chrysogenum.

The incorporation of L-[U-14C]valine into delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a direct biosynthetic precursor of penicillins and cephalosporins, was studied. When DL-methionine was added to Acremonium chrysogenum culture broths, no labeled ACV was found, while a large amount of radioactive ACV was detected when methionine was not present. DL-Norleucine, a nonsulfur analog of methionine, also inhibited the synthesis of radioactive ACV to some degree. This effect was due to the inhibition of valine transport by methionine and norleucine.

Acremonium↗