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J M Luengo

Publications and source records attributed to J M Luengo.

At least 19 recordsLinked to original sources
PubMed2000

From a short amino acidic sequence to the complete gene.

A useful strategy directed to the isolation of a required gene with a high GC content is reported. Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification. Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette). This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems.

Amino Acid Sequence↗
PubMed2000

A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed in Escherichia coli and in the nonproteolytic species Aeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the Pseudomonas aeruginosa elastase (52% identity), Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant of A. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.

Aeromonas hydrophila↗
PubMed1999

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.

Acetyl Coenzyme A↗
PubMed1998

Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway.

The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced. It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA. The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene. The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA). The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation. The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates. Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified. The Pa promoter is negatively controlled by the paaX gene product. As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative. The aerobic catabolism of PA in E. coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.

Acetyl Coenzyme A↗
PubMed1998

Elucidation of conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1.

Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45x the concentration of FeSO4 (1.8 mM) and doubling the concentration of alpha-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO4, KCl, and DTT, important in cell-free expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases.

Adenosine Triphosphate↗
PubMed1998

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon.

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

Acetyl Coenzyme A↗
PubMed1997

The phenylacetic acid uptake system of Aspergillus nidulans is under a creA-independent model of catabolic repression which seems to be mediated by acetyl-CoA.

The filamentous fungus Aspergillus nidulans is able to grow on phenylacetic acid (PhAc) as the sole carbon source and has a highly specific phenylacetic acid transport system mediating the uptake of this aromatic compound. This transport system is also able to transport some phenoxyacetic acid (PhOAc), although less efficiently. Maximal uptake rates were observed at 37 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, uptake was linear for at least 1 minute, with K(m) values for PhAc and PhOAc of 74 and 425 microM, respectively. The PhAc transport system is strongly induced by PhAc and, to a lesser extent by PhOAc and other phenyl derivatives. The utilization of glucose (and other sugars), glycerol or acetate results in a substantially reduced uptake. This negative effect caused by certain carbon sources is independent of the creA gene, the regulatory gene mediating carbon catabolite repression. Negative regulation by acetate is prevented by a loss-of-function mutation in the gene encoding acetyl-CoA synthetase, strongly suggesting that this regulation is mediated by the intracellular pool of acetyl-CoA.

Acetic Acid↗
PubMed1996

Molecular cloning and expression in different microbes of the DNA encoding Pseudomonas putida U phenylacetyl-CoA ligase. Use of this gene to improve the rate of benzylpenicillin biosynthesis in Penicillium chrysogenum.

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.

Adenosine Monophosphate↗
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↗