The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Study of the 3-Hydroxy Eicosanoyl-Coenzyme A Dehydratase and (E)-2,3 Enoyl-Coenzyme A Reductase Involved in Acyl-Coenzyme A Elongation in Etiolated Leek Seedlings1

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg−1 h−1, which is the highest ever reported for a plant system.

Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O2 uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.

The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: Molecular cloning and expression of a novel cyclooxygenase

The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase–PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and ≈50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as by-products. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

La tagatose-1,6-bisphosphate aldolase et la fructose-1,6-bisphosphate aldolase de classe I : mécanisme et stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase qui fait preuve d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-bisphosphate (TBP), mais également du fructose-1,6-bisphosphate (FBP), du sorbose-1,6-bisphosphate et du psicose-1,6-bisphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Aldolase de classe I, qui donc catalyse sa réaction en formant un intermédiaire covalent obligatoire, ou base de Schiff, avec son susbtrat, la TBP aldolase de S. pyogenes partage 14 % d’identité avec l’enzyme modèle de cette famille, la FBP aldolase de muscle de mammifère. Bien que le mécanime catalytique de la FBP aldolase des mammifères ait été examiné en détails et qu’il soit approprié d’en tirer des renseignements quant à celui de la TBP aldolase, le manque singulier de stéréospécificité de cette dernière tant dans le sens du clivage que celui de la condensation n’est toujours pas éclairci. Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution des structures de l’enzyme native et mutée, en complexe avec des subtrats et des inhibiteurs compétitifs, à des résolutions comprises entre 1.8 Å et 2.5 Å. Le trempage des cristaux de TBP aldolase native et mutante dans une solution saturante de FBP ou TBP a en outre permis de piéger un authentique intermédiaire covalent lié à la Lys205, la lysine catalytique. La determination des profils pH de la TBP aldolase native et mutée, entreprise afin d'évaluer l’influence du pH sur la réaction de clivage du FBP et TBP et ìdentifier le(s) résidu(s) impliqué(s), en conjonction avec les données structurales apportées par la cristallographie, ont permis d’identifier sans équivoque Glu163 comme résidu responsable du clivage. En effet, le mode de liaison sensiblement différent des ligands utilisés selon la stéréochimie en leur C3 et C4 permet à Glu163, équivalent à Glu187 dans la FBP aldolase de classe I, d’abstraire le proton sur l’hydroxyle du C4 et ainsi d’amorcer le clivage du lien C3-C4. L’étude du mécanimse inverse, celui de la condensation, grâce par exemple à la structure de l’enzyme native en complexe avec ses substrats à trois carbones le DHAP et le G3P, a en outre permis d’identifier un isomérisme du substrat G3P comme possible cause de la synthèse des isomères en C4 par cette enzyme. Ce résultat, ainsi que la decouverte d’un possible isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP, identifié précedemment, permet de cerner presque complètement les particularités du mécanisme de cette enzyme et d’expliquer comment elle est capable de synthétiser les quatres stéréoisomères 3(S/R), 4(S/R). De plus, la résolution de ces structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. Enfin, la résolution de la structure du mutant Lys229→Met de la FBP aldolase de muscle en complexe avec la forme cyclique du FBP, de même que des études cristallographiques sur le mutant équivalent Lys205→Met de la TBP aldolase de S. pyogenes et des expériences de calorimétrie ont permis d’identifier deux résidus particuliers, Ala31 et Asp33 chez la FBP aldolase, comme possible cause de la discrimination de cette enzyme contre les substrats 3(R) et 4(S), et ce par encombrement stérique des substrats cycliques. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

Exploring the potential of xenobiotic-metabolising enzymes as biocatalysts: Evolving designer catalysts from polyfunctional cytochrome P450 enzymes

1. Biological catalysts have the advantage of being able to catalyse chemical reactions with an often exquisite degree of regio- and stereospecificity in contrast with traditional methods of organic synthesis. 2. The cytochrome P450 enzymes involved in human drug metabolism are ideal starting materials for the development of designer biocatalysts by virtue of their catalytic versatility and extreme substrate diversity. Applications can be envisaged in fine chemical synthesis, such as in the pharmaceutical industry and bioremediation. 3. A variety of techniques of enzyme engineering are currently being applied to P450 enzymes to explore their catalytic potential. Although most studies to date have been performed with bacterial P450s, reports are now emerging of work with mammalian forms of the enzymes. 4. The present minireview will explore the rationale and general techniques for redesigning P450s, review the results obtained to date with xenobiotic-metabolising forms and discuss strategies to overcome some of the logistic problems limiting the full exploitation of these enzymes as industrial-scale biocatalysts.

Delivery of phosphonoformate prodrugs

AIDS dementia complex is a common neurological syndrome thought to result from the invasion of the CNS by HIV. Phosphonoformate has anti-HIV activity but due to its charged nature is excluded from the CNS by the blood-brain barrier. Lipophilic triesters of phosphonoformate designed to improve transport properties are unsuitable prodrugs due to their rapid and complicated hydrolysis, involving competitive P-O and P-C bond cleavage. Diesters, though hydrolytically stable, are considered too polar to passively diffuse into the CNS. Hydrophilic drugs mimicking endogenous nutrients are known to be actively transported across the blood-brain barrier. In this thesis the possibility that diesters of phosphonoformate may be actively transported is investigated. Triesters of phosphonoformate with labile aryl carboxyl esterrs were synthesised and their hydrolysis followed by 31P NMR spectroscopy. The triesters were found to undergo rapid hydrolysis via P-C bond cleavage to the phosphite. Phosphonoformate diesters designed to be analogues of actively transported -keto acids have been synthesised and fully characterised. Tyrosine-phosphonoformate and lipid-phosphonoformate conjugates have also been synthesised and characterised. An in vitro model of the blood-brain barrier utilising confluent monolayers of porcine brain microvessel endothelial cells grown on a permeable support has been established. The presence of enzyme and antigen markers specific to the blood-brain barrier has been demonstrated for the endothelial cells and the diffusional properties of the model investigated with hydrophilic and lipophilic compounds. Active transport systems for -keto acids and large amino acids have been identified in the endothelial cell monolayers using 14C-pyruvate and 3H-L-tyrosine respectively. Temperature and concentration dependence of the two systems have been demonstrated and transport constants calculated. Competition with 14C-pyruvate transport was shown with other monocarboxylic acids including the anti-epileptic drug valproate. Stereospecificity was shown in that L-lactate inhibited pyruvate transport while D-lactate did not. Sodium methyl methoxycarbonylphosphonate, a phosphonoformate diester was shown not to compete for 14C-pyruvate transport indicating that this compound has no affinity for the carrier. Competition with 3H-L-tyrosine transport was shown with other large amino acids, including the anti-Parkinsonian agent L-dopa. Stereospecificity was shown using L- and D-tyrosine and L- and D-dopa. The tyrosine-phosphonoformate conjugate, which was stable under the experimental conditions, was shown to compete with 3H-Ltyrosine transport indicating that it may be actively transported at the blood-brain barrier. Thirty two triesters, diesters and monoesters of phosphonoformate, showed no activity in an anti-HIV screen above that attributable to hydrolysis to the parent compound.