Vignette d'image

Neier, Reinhard

Nom
Neier, Reinhard
Affiliation principale
Fonction
Professeure ordinaire
Email
Reinhard.Neier@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/383

Résultat de la recherche

Filtres

6
6
6
Réinitialiser les filtres

Paramètres

Voici les éléments 1 - 6 sur 6
  • Publication
    Métadonnées seulement
    Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid
    (2002)
    Jaffe, Eileen
    ;
    Kervinen, Jukka
    ;
    Martins, Jacob
    ;
    Stauffer, Frédéric
    ;
    Neier, Reinhard 
    ;
    Wlodawer, Alexander
    ;
    Zdanov, Alexander
    Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.
  • Publication
    Métadonnées seulement
    Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism
    (2002)
    Frere, Frederic
    ;
    Schubert, Wolf-Dieter
    ;
    Stauffer, Frédéric
    ;
    Frankenberg, Nicole
    ;
    Neier, Reinhard 
    ;
    Jahn, Dieter
    ;
    Heinz, Dirk
    All natural tetrapyrroles, including hemes, chlorophylls and vitamin B-12, share porphobilinogen (PBG) as a common precursor. Porphobilinogen synthase (PBGS) synthesizes PBG through the asymmetric condensation of two molecules of aminolevulinic acid (ALA). Crystal structures of PBGS from various sources confirm the presence of two distinct binding sites for each ALA molecule, termed A and P. We have solved the structure of the active-site variant D139N of the Mg2+-dependent PBGS from Pseudomonas aeruginosa in complex with the inhibitor 5-fluorolevulinic acid at high resolution. Uniquely, full occupancy of both substrate binding sites each by a single substrate-like molecule was observed. Both inhibitor molecules are covalently bound to two conserved, active-site lysine residues, Lys205 and Lys260, through Schiff bases. The active site now also contains a monovalent cation that may critically enhance enzymatic activity. Based on these structural data, we postulate a catalytic mechanism for P. aeruginosa PBGS initiated by a C-C bond formation between A and P-side ALA, followed by the formation of the intersubstrate Schiff base yielding the product PBG. (C) 2002 Elsevier Science Ltd. All rights reserved.
  • Publication
    Métadonnées seulement
    Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity
    (2001)
    Kervinen, Jukka
    ;
    Jaffe, Eileen
    ;
    Stauffer, Frédéric
    ;
    Neier, Reinhard 
    ;
    Wlodawer, Alexander
    ;
    Zdanov, Alexander
    4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin Bit, and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-amino-levulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC50 values vary from 1 muM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn2+ are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 Angstrom resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDE code 1I8J) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved "perturbing" Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites.
  • Publication
    Métadonnées seulement
    Inhibition studies of porphobilinogen synthase from Escherichia coli differentiating between the two recognition sites
    (2001)
    Stauffer, Frédéric
    ;
    Zizzari, Eleonor
    ;
    Jarret, Caroline
    ;
    Faurite, Jean-Philippe
    ;
    Bobalova, Janette
    ;
    Neier, Reinhard 
    Porphobilinogen synthase condenses two molecules of 5-amino-levulinate in an asymmetric way. This unusual transformation requires a selective recognition and differentiation between the :substrates ending up in the A site or in the P site of porphobilinogen synthase. Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites. The P site, whose structure is known from X-ray crystallographic studies, tolerates ester functions well. The A site interacts very strongly with nitro groups, but is not very tolerant to ester functions. This differentiation is a central factor in the asymmetric I handling of the two identical substrates. Finally, it could be shown nor the keto group of-the,Substrate bound at the A site is not Only essential for the recognition, but that an increase in electrophilicity of-the carbon atom also increases the inhibition potency considerably. This has important consequences for the recognition process at the A site, whose-exact structure is not yet known.
  • Publication
    Métadonnées seulement
    Porphobilinogen synthase: A challenge for the chemist?
    (2001)
    Stauffer, Frédéric
    ;
    Zizzari, Eleonor
    ;
    Soldermann-Pissot, Carole
    ;
    Faurite, Jean-Philippe
    ;
    Neier, Reinhard 
    The initial steps in the biosynthesis of the tetrapyrrolic dyes, called the 'pigments of life', are highly convergent. The formation of porphobilinogen, the pyrrolic precursor of the tetrapyrrolic skeleton, uses delta -aminolevulinate as the starting material. This amino acid is dedicated to the biosynthesis of tetrapyrroles, However, the chemical condensation of delta -aminolevulinate leads to a symmetric pyrazine, Attempts to imitate the biosynthesis using one of the proposed pathways for the biosynthesis of porphobilinogen as a guideline has allowed us to synthesize a protected precursor of porphobilinogen in an efficient way. Based on the two major proposals for the biosynthesis, a series of specifically synthesized inhibitors was also tested. The inhibition behavior and the potency of the inhibitors expressed as their K-i value has unraveled an interesting relationship between the structure of the inhibitor and the strength of its interaction with the active site. The concerted use of mechanistic analysis, synthesis and kinetic studies of inhibitors has increased our knowledge about the enzyme porphobilinogen synthase, Structural studies of enzyme-inhibitor complexes will hopefully complement the kinetic results accumulated so far.
  • Publication
    Métadonnées seulement
    Tandem nucleophilic addition/Diels-Alder reaction of N-butadienyl N,O-ketene silyl acetals with C-60: Stereoselective formation of bicyclic octahydroquinoline-1,2,3,4-tetrahydrobuckminsterfullerenes and combined NMR spectroscopic and computational evaluation of the functionalization reactions
    (1999)
    Rubin, Yves
    ;
    Ganapathi, Padma
    ;
    Franz, Andreas
    ;
    An, Yi-Zhong
    ;
    Qian, Wenyuan
    ;
    Neier, Reinhard 
    We have studied the reactivity of the N,O-ketene N-1,3-butadienyl-N-alkyl-O-silyl acetals 1a-e with C-60 proceeding through a tandem process to give the adducts 2a-e, The addition order of these tandem reactions has been evaluated. The initial nucleophilic Michael-like addition of the electron-rich N,O-ketene acetal moiety proceeds unusually fast at 25 degrees C, followed by an intramolecularly accelerated Diels-Alder step that is highly diastereoselective. The structures of compounds 2a-e were determined from the H-1 and C-13 NMR shifts and from II-II coupling patterns, while their stereochemistry was deduced from 2D T-ROESY NMR experiments. The proposed mechanism for the nucleophilic addition involves single electron transfer followed by radical anion-radical cation recombination. Computational investigations of the reaction pathways, transition states, and conformational energies have been carried out to corroborate the experimental data.