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J M Luengo

Publications and source records attributed to J M Luengo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Enzymatic synthesis of hydrophobic penicillins.

Our knowledge of the enzymes and genes involved in the biosynthesis of beta-lactam antibiotics has increased notably in the last decade. The purification to homogeneity of some of these proteins as well as their biochemical characterization has allowed some of them to be used for synthesizing many different penicillins and cephalosporin-like products in vitro. In this report we describe the most important advances in this field, placing special emphasis on the enzymatic synthesis of hydrophobic penicillins. The use of purified acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum and several acyl-CoA ligases obtained from different microbial origins has led to the reproduction "in vitro" of the last step involved in in penicillin biosynthesis. By coupling these enzymatic systems (AT and acyl-CoA ligases) an impressive number of beta-lactam antibiotics has been obtained. Thus, most of the known natural penicillins, many of the semisynthetic variants and others, which until now can only be obtained chemically, have been synthesized enzymatically from their natural precursors. Furthermore, the use of heterologous proteins in coupled systems has opened a new and exciting field in beta-lactam antibiotic research, lending new perspectives to the traditional methodology followed by antibiotic fermentation industries.

Acyl Coenzyme A↗
PubMed1994

Catabolism of aromatics in Pseudomonas putida U. Formal evidence that phenylacetic acid and 4-hydroxyphenylacetic acid are catabolized by two unrelated pathways.

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.

Chromatography, High Pressure Liquid↗
PubMed1994

Inhibition of penicillin biosynthetic enzymes by halogen derivatives of phenylacetic acid.

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.

Acyltransferases↗
PubMed1994

Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate.

The phenylacetic acid transport system (PATS) of Pseudomonas putida U was studied after this bacterium was cultured in a chemically defined medium containing phenylacetic acid (PA) as the sole carbon source. Kinetic measurement was carried out, in vivo, at 30 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, the uptake rate was linear for at least 3 min and the value of Km was 13 microM. The PATS is an active transport system that is strongly inhibited by 2,4-dinitrophenol, 4-nitrophenol (100%), KCN (97%), 2-nitrophenol (90%), or NaN3 (80%) added at a 1 mM final concentration (each). Glucose or D-lactate (10 mM each) increases the PATS in starved cells (140%), whereas arsenate (20 mM), NaF, or N,N'-dicyclohexylcarbodiimide (1 mM) did not cause any effect. Furthermore, the PATS is insensitive to osmotic shock. These data strongly suggest that the energy for the PATS is derived only from an electron transport system which causes an energy-rich membrane state. The thiol-containing compounds mercaptoethanol, glutathione, and dithiothreitol have no significant effect on the PATS, whereas thiol-modifying reagents such as N-ethylmaleimide and iodoacetate strongly inhibit uptake (100 and 93%, respectively). Molecular analogs of PA with a substitution (i) on the ring or (ii) on the acetyl moiety or those containing (iii) a different ring but keeping the acetyl moiety constant inhibit uptake to different extents. None of the compounds tested significantly increase the PA uptake rate except adipic acid, which greatly stimulates it (163%). The PATS is induced by PA and also, gratuitously, by some phenyl derivatives containing an even number of carbon atoms on the aliphatic moiety (4-phenyl-butyric, 6-phenylhexanoic, and 8-phenyloctanoic acids). However, similar compounds with an odd number of carbon atoms (benzoic, 3-phenylpropionic, 5-phenylvaleric, 7-phenylheptanoic, and 9-phenylnonanoic acids) as well as many other PA derivatives do not induce the system, suggesting that the true inducer molecule is phenylacetyl-coenzyme A (PA-CoA). Furthermore, after P. putida U is cultured in the same medium containing other carbon sources (glucose or octanoic, benzoic, or 4-hydroxyphenylacetic acid) in the place of PA, the PATS and PA-CoA are not detected; neither the PATS nor PA-CoA is found in cases in which mutants (PA- and PCL-) lacking the enzyme which catalyzed the initial step of the PA degradation (phenylacetyl-CoA ligase) are used. PA-CoA has been extracted from bacteria and identified as a true PA catabolite by high-performance liquid chromatography and also enzymatically with pure acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum.

Acetyl Coenzyme A↗
PubMed1993

Characterisation of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes.

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.

Acetate-CoA Ligase↗
PubMed1993

Polysialic acids.

1. Polysialic acids are linear homopolymers of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminated neuraminic acid (KDN) residues joined by alpha 2,8, alpha 2-9 or alpha 2,8/alpha 2,9 ketosidic linkages. 2. They occur in glycoproteins of embryonic neural membranes (playing a role of neural cell adhesion molecules), in non-neural tissues (postnatal kidney), tumours, (neuroectodermal tumours), fish eggs and in the capsule of certain bacteria such as Neisseria meningitidis group B. 3. These polymers are synthesized through reactions which involve (a) the synthesis of sialic acid; (b) its activation to a cytidine monophosphate sugar nucleotide and (c) the polymerization of the different residues by a polysialyl-transferase complex. 4. Polysialic acids are involved in organogenesis and in cell growth. In several tissues they act as oncodevelopmental antigens, and in bacteria are also virulent determinants.

Animals↗
PubMed1993

Comparative analysis of the antibodies against capsular polysaccharides of Escherichia coli K-92 and K-235: an immunochemical method for the identification of polysialic acids.

1. The antibodies produced against the capsular poly-N-acetylneuraminic acid (poly-Neu5Ac) of E. coli K-92 (alpha 2-8-, alpha 2-9-linked) were 100-fold less sensitive than those obtained against E. coli K-235 capsular polysaccharide (CP) (alpha 2-8-linked) and recognized both kinds of polymers to a similar extent. 2. The partial hydrolysis of each purified polysaccharide revealed that E. coli K-92 CP is more labile at acidic pH than the polymer alpha 2-8-linked of E. coli K-235. 3. The antisera against CP from E. coli K-92 bound its own oligomers in which the number of Neu5Ac units was higher than three, whereas they only cross-reacted with the oligomers derived from E. coli K-235 containing a number of residues higher than 12. 4. The antisera against E. coli K-235 CP that recognized alpha 2-8 oligomers with a number of Neu5Ac residues higher than 5, also reacted, although very weakly, with those containing alpha 2-8 and alpha 2-9 linkages in which the carbon length was higher than (Neu5Ac)3. 5. Both types of antibodies were also able to recognize the native antigens in living bacteria and could be employed for the recognition of the type of linkage presents in different sialylpolymers.

Antibodies, Bacterial↗
PubMed1993

Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

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.

Caprylates↗
PubMed1992

Use of long-chain fatty acid-CoA ligase (AMP-forming) from Pseudomonas fragi for the 'in vitro' synthesis of natural penicillins.

Five different naturally occurring penicillins containing as side chains hexanoic, trans-3-hexenoic, heptanoic, octanoic or trans-3-octenoic acids have been synthesized 'in vitro' by coupling long-chain fatty acid-CoA ligase (AMP-forming) (EC 6.2.1.3) from Pseudomonas fragi (LFCoA-L) with acyl-CoA: 6-aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. The quantity of penicillin produced was directly related with the carbon length of the side chain precursor tested, being maximal with octanoic acid. Fatty acids with a lower length than C5 were not recognized as substrates and nor were certain aromatic molecules.

Acyl Coenzyme A↗
PubMed1992

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

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.

Acetate-CoA Ligase↗
PubMed1992

Characterization of an inducible transport system for glycerol in Streptomyces clavuligerus. Repression by L-serine.

Streptomyces clavuligerus NRRL 3585 grown in a chemically defined medium containing glycerol as the sole carbon source transported this molecule by two different systems. One of these was constitutive with a very low uptake efficiency and insufficient to attend to the metabolic requirements of this bacterium (constitutive glycerol transport system) and the other (glycerol transport system (GTS)) active and specifically induced by D-glycerol which is responsible for the transport of more than 90% of the glycerol taken up the cells. GTS was seen to have an optimal pH and temperature of 7.0 and 30 degrees C, respectively, and its Km was 14 microM. It was repressed by L-serine and addition of this amino acid to the culture broth (10 mM) inhibited the growth of S. clavuligerus but not that of other species of Streptomyces.

Biological Transport, Active↗
PubMed1991

H.p.l.c. of oligo(sialic acids). Application to the determination of the minimal chain length serving as exogenous acceptor in the enzymic synthesis of colominic acid.

A rapid, sensitive and easy h.p.l.c. method was developed for the quantitative analysis of oligosialic acids. This procedure which permits the complete separation (in 23 min) of several sialyloligomers with a degree of polymerization of between 1 and 16, has been employed to establish the minimal chain length of oligomer accepted, as an exogenous acceptor, by Escherichia coli K-235 sialytransferase complex (ST) leading to the synthesis in vitro of colominic acid. We showed that this membrane-bound enzyme catalyses the direct transfer of Neu5Ac residues (one by one) from CMP-Neu5Ac to an exogenous acceptor molecule which contains at least three Neu5Ac residues. Free Neu5Ac or (Neu5Ac)2 were not recognized as substrates, whereas the maximal rate of polymer elongation was achieved when (Neu5Ac)5 was used as substrate.

Chromatography, High Pressure Liquid↗
PubMed1991

Synthesis of 3-furylmethylpenicillin using an enzymatic procedure.

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.

Acyltransferases↗
PubMed1991

Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: a very sensitive assay for a newly described enzyme.

Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.

Acetyl Coenzyme A↗
PubMed1991

In vitro enzymatic synthesis of new penicillins containing keto acids as side chains.

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.

Keto Acids↗
PubMed1991

Acyl-CoA: 6-APA acyltransferase from Penicillium chrysogenum studies on its hydrolytic activity.

Acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 catalyzes the hydrolysis of different acyl-CoA derivatives generating, in the absence of 6-APA, free acid and CoA. The hydrolytic efficiency of AT is highest for acyl-CoA variants in which the acyl-moiety is higher than six carbon atoms. The maximal rate of catalysis was achieved in 50 mM Tris-HCl buffer, pH 8.5 at 35 degrees C. Unlike the AT activity, the acylase activity has a different optimum temperature and substrate specificity and dithiothreitol is not required for the reaction.

Acyl Coenzyme A↗