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Neier, Reinhard

Nom
Neier, Reinhard
Affiliation principale
Fonction
Professeure ordinaire
Email
Reinhard.Neier@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/383
_
0000-0003-0890-1797

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  • 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
    Accès libre
    Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic
    (2002)
    Jaffe, Eileen K.
    ;
    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
    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.