Showing papers by "Antoni R. Slabas published in 1996"

A mechanism of drug action revealed by structural studies of enoyl reductase.

Abstract: Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2' hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.

Towards the genetic engineering of triacylglycerols of defined fatty acid composition: major changes in erucic acid content at the sn-2 position affected by the introduction of a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii into oil seed rape

Abstract: A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.

Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus : cloning and analysis of expression during oilseed rape embryogenesis

Abstract: In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were clones, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the beta-carboxyltransferase subunit (betaCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and betaCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

Cloning of Brassica napus CTP: phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant.

Abstract: CTP:phosphocholine cytidylyltransferase is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. We have isolated four cDNAs for the cytidylyltransferase from a root cDNA library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequences of the B. napus enzymes resembled rat and yeast enzymes in the central domain. Although all cytidylyltransferases ever cloned from B. napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B. napus cDNA clones restored the cytidylyltransferase activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast cytidylyltransferase to membranes, and may indicate that the B. napus enzyme was compatible with the machinery.

Dna sequence encoding plant 2-acyltransferase

Abstract: Plants, particularly transgenic plants, may be produced having a 2-acyltransferase enzyme from Limnanthes with an altered substrate specificity compared to the native enzyme. For example, oil seed rape Brassica napus may contain the 2-acyltransferase transgene derived from Limnanthes douglasii in order to produce trierucin. The cDNA sequence of Limnanthes douglasii 2-acyltransferase and its equivalent protein sequence are disclosed.

Crystallization of Escherichia coli enoyl reductase and its complex with diazaborine.

Abstract: Recent work has shown that the NADH-dependent enoyl acyl carrier protein reductase from Escherichia coli is the target for diazaborine, an antibacterial agent. This enzyme has been crystallized by the hanging-drop method of vapour diffusion complexed with NAD+ and in the presence and absence of a thieno diazaborine. The crystals grown in the absence of diazaborine (form A) are in the space group P21 with unit-cell dimensions a = 74.0, b = 81.2, c = 79.0 A and β = 92.9°, and with a tetramer in the asymmetric unit, whilst those grown in the presence of diazaborine (form B) are in the space group P6122 (or P6522) with unit-cell dimensions a = b = 80.9 and c = 328.3 A, and with a dimer in the asymmetric unit. The structure determination of this enzyme in the presence of diazaborine will provide information on the nature of the drug binding site and contribute to a programme of rational drug design.

Soluble and membrane bound components of plant lipid synthesis

Abstract: L'enoyl ACP reductase (ENR) catalyse la reduction NADH-dependante du trans enoyl ACP pour former les acyl ACP satures ; il s'agit d'un composant essentiel de l'acide gras synthetase de type Il fortement exprimee d'une facon temporelle dans les semences. L'enzyme a ete purifiee a partir du colza, entierement sequencee, son ADNc clone, et la proteine surexprimee et cristallisee. La structure tridimensionnelle complete de l'enzyme a ete determinee a 1,9 A. Le crotonyl ACP est un meilleur substrat que le crotonyl CoA car ce dernier se fixe egalement au site du NADH, se comportant ainsi comme un inhibiteur de l'enzyme. Le site actif potentiel a ete identifie a partir des acides amines conserves et de la position du noyau nicotinamide du NADH. De plus, des analogies structurales ont ete trouvees entre l'enoyl ACP reductase et la 3α-, 20β-hydroxysteroide deshydrogenase. Cela nous a donne des renseignements importants sur le mecanisme de la reaction qui seront testes par mutagenese dirigee. Dans le but d'etudier les enzymes membranaires impliquees dans la synthese des lipides, nous avons utilise la technique du clonage par complementation dans E. coli pour isoler la 2-acyltransferase liee aux membranes qui n'a jamais pu etre isolee par les procedes traditionnels. En premier lieu, nous avons clone une 2-acyltransferase a partir du mais et plus recemment nous avons clone deux 2-acyltransferases a partir de Limnanthes douglasii. L'une des ces enzymes presente des caracteristiques fonctionnelles distinctes de la 2-acyltransferase de E. coli. L'introduction d'un ADNc codant pour la 2-acyltransferase dans une souche de colza produisant de l'acide erucique nous a permis de synthetiser la trierucine dans les semences transgeniques.

Sequence d'adn codant pour de la 2-acyltransferase vegetale

Abstract: Il est possible de produire des plantes, et en particulier des plantes transgeniques presentant une enzyme du type 2-acyltransferase a partir de Limnanthes presentant une specificite alteree du substrat par comparaison avec l'enzyme originale Par exemple la graine de colza oleagineux Brassica napus peut contenir les deux transgenes de 2-acyltransferase derivant du Limnanthes douglasii permettant de produire de la trierucine La sequence d'ADNc de la 2-acyltransferase du Limnanthes douglasii et sa sequence proteique equivalente sont presentees ici