Structure of Porphobilinogen Synthase from Pseudomonas aeruginosa in Complex with 5-Fluorolevulinic Acid Suggests a Double Schiff Base Mechanism

Frere, F.; Schubert, Wolf‐Dieter; Stauffer, F.; Frankenberg, Nicole; Neier, Reinhard; Jahn, Dieter; Heinz, Dirk W.

doi:10.1016/s0022-2836(02)00472-2

Cited by 55 publications

(48 citation statements)

References 29 publications

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“…The crystal structure of ALAD from P. aeruginosa reveals that Mg 2ϩ is bound 14 Å away from the Schiff base-forming nitrogen atom of the active site lysine (16). Based on the crystal structure of the enzyme in complex with the inhibitor 5-fluorolevulinic acid at high resolution, the active site has been shown to contain a monovalent cation (Na ϩ ) that may assist in enzyme activity (17). rPfALAD is similar to PsALAD in this regard and added K ϩ stimulates the enzyme activity in the pH range between 6.5 and 7.5.…”

Section: Discussionmentioning

confidence: 99%

“…The enzymatically active preparation was used to measure the intrinsic Mg 2ϩ content. PfALAD Homology Modeling-Alignment of the sequence (accession number CAC82990/Q8MYK5) of PfALAD with the sequence neighbors identified by a BLAST (14) search of the Non-Redundant and Protein Data Bank (15) data bases, indicated ALAD from P. aeruginosa (PsALAD, Protein Data Bank codes 1B4K and 1GZG) (16,17) to be the closest structural template with sequence similarities of 56% (E value: 3e Ϫ50 ) over 330 amino acid residues. The alignment with the template, when carefully verified by examining additional pairwise (18) and multiple alignments (19) with other closely related sequences, indicated a few local frameshifts as judged by the position of well conserved known functional residues, which were then corrected by minor editing.…”

Section: Determination Of Mg 2ϩmentioning

confidence: 99%

“…A series of events leading to the formation of a fully assembled active site (monomer A in dimer AB) has been proposed for PsALAD (17). These are (a) entry of monovalent cation to the catalytic center that is then stabilized by is altered slightly because of the influence of an insertion at position 205 and a change in the amino acid sequence from position 218 to 221 in PfALAD; (b) the two adjacent residues, Asn 315 and Gln 317 , form bidentate hydrogen bonds in PfALAD that are absent in PsALAD; and (c) His 223 , which is in the vicinity of Lys 316 , keeps the Lys 316 fixed and oriented (to avoid charge repulsion) toward the substrate.…”

Section: Modeling Of Pfalad Structurementioning

confidence: 99%

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δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Dhanasekaran

Chandra

Sagar

et al. 2004

Journal of Biological Chemistry

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The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host ␦-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit M r of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20 -30% upon addition of Mg 2؉ . The enzyme is inhibited by Zn 2؉ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg 2؉ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.Studies in this laboratory had demonstrated that the malaria parasites (Plasmodium falciparum and Plasmodium berghei) are capable of heme biosynthesis de novo, despite acquiring large amounts of heme from the host red cell hemoglobin in the intraerythrocytic stage (1, 2). Inhibition of the de novo heme biosynthetic pathway leads to death of the parasite and the pathway is therefore a drug target (1-3). Studies on the enzymes of the heme-biosynthetic pathway have revealed that the first enzyme, ␦-aminolevulinate synthase, is coded for by the parasite genome and is localized in the parasite mitochondrion (4, 5). Studies from this laboratory have shown that the second enzyme of the pathway, ␦-aminolevulinate dehydratase (ALAD), 1 in the parasite is of host origin and evidence was provided to show that the enzyme is translocated (imported) from the host red cell into the parasite and is functional (2, 3).At the same time genome sequence data available for P. falciparum indicates that the parasite genome codes for ALAD (PfALAD) and other enzymes of the heme-biosynthetic pathway. Sato et al. (6) have shown by phylogenetic amino acid sequence analysis derived from truncated...

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Section: Discussionmentioning

confidence: 99%

Section: Determination Of Mg 2ϩmentioning

confidence: 99%

Section: Modeling Of Pfalad Structurementioning

confidence: 99%

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δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Dhanasekaran

Chandra

Sagar

et al. 2004

Journal of Biological Chemistry

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“…Recently, the structure determination of the complex between PBGS and 5-fluorolevulinic acid (6) showed that two identical substrate analogs may bind at the Scheme 1 same time to the active site [23]. This X-ray structure provides a detailed model of the native substrate -enzyme complex and fully identifies all residues involved in A-site as well as P-site formation for Zn-independent PBGS.…”

mentioning

confidence: 88%

“…This X-ray structure provides a detailed model of the native substrate -enzyme complex and fully identifies all residues involved in A-site as well as P-site formation for Zn-independent PBGS. It reveals that an alkaline metal ion may be required in Mg-dependant PBGS to stabilize ALA (1) in a defined conformation in the polar A-site and modulate the activity of P-side ALA (1) [23].…”

mentioning

confidence: 99%

Synthesis of Bisubstrate Inhibitors of Porphobilinogen Synthase from Pseudomonas aeruginosa

Gacond

Frere

Nentwich

et al. 2007

Chemistry & Biodiversity

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Porphobilinogen synthase (PBGS) synthesizes porphobilinogen 2 (PBG), the common precursor of all natural tetrapyrroles, through an asymmetric condensation of two molecules of 5-aminolevulinic acid 1 (ALA). Symmetrically linked dimers 7-11 derived from levulinic acid 3 (gamma-oxovaleric acid) have been synthesized to mimic the assumed bisubstrate bound to the active site of the enzyme. Their inhibition potential was characterized by determination of the IC(50) and K(i) values using PBGS from Pseudomonas aeruginosa. The polarity and the size of the functional group linking the two levulinic acid 3 units have a strong influence on the inhibition behavior.

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5‐Aminolaevulinic Acid Dehydratase

Cooper

Erskine

2004

Handbook of Metalloproteins

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The enzyme 5‐aminolaevulinic acid dehydratase (ALAD, E.C.4.2.1.24), sometimes referred to as porphobilinogen synthase, catalyzes the second step in the biosynthesis of tetrapyrroles involving the condensation between two 5‐aminolaevulinic acid (ALA) molecules to form the pyrrole porphobilinogen (PBG). The X‐ray structures of several ALADs have been determined showing that the enzyme forms a large homo‐octameric structure with all eight active sites on the outer surface. Each subunit adopts the triose phosphate isomerase (TIM) barrel fold with an N‐terminal arm, which forms extensive intersubunit interactions. Pairs of monomers associate with their arms wrapped around each other to form compact dimers. Four dimers, which interact principally via their arm regions, form the octamer, which has a large solvent‐filled cavity in the center. The active site of each subunit is located in a pronounced cavity formed by loops at the C‐terminal ends of the strands forming the TIM barrel. In humans, hereditary deficiencies in ALAD give rise to the rare disease Doss porphyria, and the sensitivity of the enzyme to inhibition by lead ions is thought to be one of the main problems in acute lead poisoning. ALADs from many species (except plants and some bacteria) have a zinc ion at the active site, which has been implicated in the catalytic mechanism and is readily displaced by the inhibitor lead. These ALADs have a cysteine‐rich motif that coordinates the active site zinc ion (α‐site). In contrast, ALADs from plants and some bacteria have a number of mutations in the metal‐binding motif that replace the cysteine residues with other amino acids. Two of the cysteines are replaced by aspartate, which is thought to be more appropriate for coordination of magnesium ions, which these enzymes apparently require instead of zinc. Some ALADs have an additional zinc or magnesium binding site located between the subunits of the octamer (β‐site) that has more of a structural or activating role. Early inhibitor binding studies defined the interactions made by only one of the two substrate moieties (P‐side substrate) that bind to the enzyme during catalysis. Biochemical and crystallographic data showed that P‐side substrate binds by forming a Schiff base with the enzyme at Lys247 ( Escherichia coli ALAD numbering). Until very recently there has been little evidence that binding of the second substrate moiety (A‐side substrate) involves formation of a Schiff base with the enzyme. However, the structures of diacid inhibitors provided an improved definition of the interactions made by both the substrate molecules (A‐ and P‐side). The most intriguing result was the finding that 4,7‐dioxosebacic acid forms a second Schiff base with the enzyme involving Lys195 ( E. coli ALAD numbering), suggesting that A‐side substrate makes the same interaction during catalysis. This finding has been substantiated by the demonstration that the inhibitor 5‐fluorolaevulinic acid binds by making Schiff bases with both the active site lysine residues. Current proposals for the catalytic mechanism involve the binding of both substrate moieties by formation of Schiff bases with the active site lysines.

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Structure of Porphobilinogen Synthase from Pseudomonas aeruginosa in Complex with 5-Fluorolevulinic Acid Suggests a Double Schiff Base Mechanism

Cited by 55 publications

References 29 publications

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Synthesis of Bisubstrate Inhibitors of Porphobilinogen Synthase from Pseudomonas aeruginosa

5‐Aminolaevulinic Acid Dehydratase

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