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Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid
The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 Å. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).
Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic
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.
Porphobilinogen synthase: A challenge for the chemist?
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.
Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates
Background: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon–carbon bond formation (an aldol-type reaction) and the second carbon–nitrogen bond formation, leading to an iminium ion.
Results: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis–Menten behavior of substrate analog inhibitors — they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior.
Conclusions: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon–nitrogen bond formation occurs first.
Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism
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.
Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity
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.
Inhibition Studies of Porphobilinogen Synthase from Escherichia coli Differentiating between the Two Recognition Si
Porphobilinogen synthase condenses two molecules of 5-aminolevulinate 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 handling of the two identical substrates. Finally, it could be shown that 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.
Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid
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.
Inhibition studies of porphobilinogen synthase from Escherichia coli differentiating between the two recognition sites
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.
The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors
The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.