The Experts below are selected from a list of 2196 Experts worldwide ranked by ideXlab platform
Eileen K Jaffe - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of toxoplasma gondii Porphobilinogen synthase insights on octameric structure and Porphobilinogen formation
Journal of Biological Chemistry, 2011Co-Authors: Eileen K Jaffe, Dhanasekaran Shanmugam, Shellie H Dieterich, Lance Stewart, Anna S. Gardberg, Banumathi Sankaran, Peter J Myler, David S RoosAbstract:Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product Porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.
-
the Porphobilinogen synthase catalyzed reaction mechanism
Bioorganic Chemistry, 2004Co-Authors: Eileen K JaffeAbstract:Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrroles, the asymmetric condensation of two molecules of δ-aminolevulinic acid to form Porphobilinogen. There is a variable requirement for an essential active site zinc that necessitates consideration of PBGS as an enzyme that may exhibit phylogenetic diversity in its chemical reaction mechanism. Recent crystal structures suggest reaction mechanisms that involve two covalent Schiff base linkages between adjacent active site lysine residues and each of the two substrate molecules. The reaction appears to stall at a covalently bound almost-product intermediate that is poised for breakdown to product upon binding of a substrate molecule to an adjacent active site and a subsequent conformational change.
-
mechanistic implications of mutations to the active site lysine of Porphobilinogen synthase
Journal of Biological Chemistry, 2001Co-Authors: Laura W Mitchell, Jacob Martins, Marina Volin, Eileen K JaffeAbstract:Abstract Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices.13C NMR studies of [3,5-13C]Porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e.environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coliPBGS and K246G demonstrate that both bind Porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.
-
Porphobilinogen synthase from pea expression from an artificial gene kinetic characterization and novel implications for subunit interactions
Biochemistry, 2000Co-Authors: Jukka Kervinen, Samuel Litwin, Jacob Martins, Marina Volin, Robert C Scarrow, And Erica Yoon, Anthony T. Yeung, Roland L Dunbrack, Eileen K JaffeAbstract:Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B12. The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor Porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pKa values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kine...
-
pseudomonas aeruginosa contains a novel type v Porphobilinogen synthase with no required catalytic metal ions
Biochemistry, 1999Co-Authors: Nicole Frankenberg, Dieter Jahn, Eileen K JaffeAbstract:: Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]Porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize Porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.
Reinhard Neier - One of the best experts on this subject based on the ideXlab platform.
-
facile synthesis of a ready to use precursor of Porphobilinogen and its amino acid derivatives
Journal of Organic Chemistry, 2008Co-Authors: Carole Pissot Soldermann, Ramakrishnan Vallinayagam, Manuel Tzouros, Reinhard NeierAbstract:A practical synthesis of Porphobilinogen based on the biosynthetic mechanism is described. The crossed Mukayiama aldol reaction is the key step creating the central carbon-carbon bond between the two protected forms of 5-aminolevulinic acids. The optimized sequence gives a crystalline, storable precursor, which can be transformed in high yield into Porphobilinogen and bioconjugates thereof. The enzymatic hydrolysis of the precursor produces Porphobilinogen in quantitative yield.
-
synthesis of bisubstrate inhibitors of Porphobilinogen synthase from pseudomonas aeruginosa
Chemistry & Biodiversity, 2007Co-Authors: S Gacond, F Frere, Jeanphilippe Faurite, Michael Nentwich, Nicole Frankenbergdinkel, Reinhard NeierAbstract:: 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.
-
probing the active site of pseudomonas aeruginosa Porphobilinogen synthase using newly developed inhibitors
Biochemistry, 2006Co-Authors: F Frere, Dirk W Heinz, S Gacond, Michael Nentwich, Reinhard Neier, Nicole FrankenbergdinkelAbstract:: Porphobilinogen synthase catalyzes the first committed step of the tetrapyrrole biosynthesis pathway. In an aldol-like condensation, two molecules of 5-aminolevulinic acid (ALA) form the first pyrrole, Porphobilinogen. Newly synthesized analogues of a reaction intermediate of Porphobilinogen synthase have been employed in studying the active site and the catalytic mechanism of this early enzyme of tetrapyrrole biosynthesis. This study combines structural and kinetic evaluation of the inhibition potency of these inhibitors. In addition, one of the determined protein structures provides for the first time structural evidence of a magnesium ion in the active site. From these results, we can corroborate an earlier postulated enzymatic mechanism that starts with formation of a C-C bond, linking C3 of the A-side ALA to C4 of the P-side ALA through an aldole addition. The obtained data are discussed with respect to the current literature.
-
inhibition of escherichia coli Porphobilinogen synthase using analogs of postulated intermediates
Chemistry & Biology, 2000Co-Authors: Caroline Jarret, Rainer Martin Luond, Frederic Stauffer, Matthias E Henz, Maurus Marty, Janette Bobalova, Peter Schürmann, Reinhard NeierAbstract:Abstract 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.
-
on the formation of the mixed pyrrole catalysed by Porphobilinogen synthase from rhodobacter spheroides
Biochimica et Biophysica Acta, 1996Co-Authors: Rainer Martin Luond, Reinhard NeierAbstract:Abstract The enzyme Porphobilinogen synthase (PBGS) catalyses the formation of Porphobilinogen (PBG) from two molecules of 5-aminolevulinic acid (ALA). It has been claimed that the PBGS from Rhodobacter spheroides is able to form a mixed pyrrole, from one molecule of 5-aminolevulinic acid and one molecule of levulinic acid. The chemical synthesis of this mixed pyrrole allowed to show, that the compound formed from 5-aminolevulinic acid and levulinic acid with PBGS from R. spheroides has not the proposed structure. The putative enzyme catalysed formation of the mixed pyrrole had been used as an argument for the postulated mechanism of PBGS. In view of our results this line of arguments has to bee re-evaluated.
Marina Volin - One of the best experts on this subject based on the ideXlab platform.
-
mechanistic implications of mutations to the active site lysine of Porphobilinogen synthase
Journal of Biological Chemistry, 2001Co-Authors: Laura W Mitchell, Jacob Martins, Marina Volin, Eileen K JaffeAbstract:Abstract Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices.13C NMR studies of [3,5-13C]Porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e.environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coliPBGS and K246G demonstrate that both bind Porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.
-
Porphobilinogen synthase from pea expression from an artificial gene kinetic characterization and novel implications for subunit interactions
Biochemistry, 2000Co-Authors: Jukka Kervinen, Samuel Litwin, Jacob Martins, Marina Volin, Robert C Scarrow, And Erica Yoon, Anthony T. Yeung, Roland L Dunbrack, Eileen K JaffeAbstract:Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B12. The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor Porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pKa values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kine...
-
crystallization and preliminary x ray diffraction studies of e coli Porphobilinogen synthase and its heavy atom derivatives
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: L Shimonilivny, H L Carrell, Kaufman A Katz, Carol E Afshar, Laura W Mitchell, Marina Volin, Eileen K Jaffe, T Wagner, Jenny P GluskerAbstract:Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form Porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported. These crystals diffract to about 3.0 A resolution.
-
5 chlorolevulinate interactions with bradyrhizobium japonicum Porphobilinogen synthase
Bioorganic Chemistry, 1995Co-Authors: Eileen K Jaffe, Robert M Petrovich, Marina VolinAbstract:Abstract 5-Chlorolevulinic acid (5-CLA) is an α-chloroketone analog of the tetrapyrrole precursor 5-aminolevulinate (ALA). As such it serves as an excellent probe of the mechanisms of enzymes which form or utilize ALA. The enzyme Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of ALA to form the monopyrrole Porphobilinogen. In principle, 5-CLA could bind at either or both of the two different ALA binding sites of PBGS. In the presence of an active-site nucleophile, 5-CLA might chemically modify the protein with concomitant inactivation. Such inactivation has been shown for mammalian and Escherichia coli PBGS, each of which has reactive cysteines at the active site. In contrast, we report here that 5-CLA does not readily inactivate Bradyrhizobium japonicum PBGS ( Bj PBGS) in either the presence or the absence of ALA. At neutral pH 5-CLA is a reversible inhibitor of Bj PBGS ( K i = 47 μM) and affects both the K m for ALA (0.8 mM) and the V max of the reaction. Bj PBGS does not use 5-CLA as an alternative substrate to form the 11-chloro analog of Porphobilinogen in a mixed condensation with ALA. 13 C NMR was used to observe 5-chloro-[1,4- 13 C]levulinate bound at the Bj PBGS active site. The 13 C NMR chemical shifts indicate that 5-CLA preferentially binds to the first ALA binding site and undergoes formation of an analog of the first Schiff base intermediate which normally occurs in the PBGS-catalyzed reaction. These data contribute to a growing body of mechanistic work which supports the proposition that carbon-nitrogen bond formation precedes carbon-carbon bond formation in the PBGS-catalyzed biosynthesis of Porphobilinogen.
Frantisek Turecek - One of the best experts on this subject based on the ideXlab platform.
-
direct assay of δ aminolevulinic acid dehydratase in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry
Analytical Chemistry, 2010Co-Authors: John Choiniere, Ronald C Scott, Michael H Gelb, Frantisek TurecekAbstract:We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into Porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of Porphobilinogen to its butyramide, and liquid−liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated Porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The Km of ALAD for ALA was measured as 333 μM, and the Vmax was 19.3 μM/h. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μ...
-
direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry Porphobilinogen deaminase
Analytical Chemistry, 2008Co-Authors: Yuesong Wang, Ronald C Scott, Michael H Gelb, Frantisek TurecekAbstract:We report a new assay of human Porphobilinogen deaminase (PBGD). Deficiency in this enzyme activity causes acute intermittent porphyria, the most common disorder of heme biosynthesis. The assay involves incubation of blood erythrocyte lysate with Porphobilinogen, the natural PBGD substrate. Two subsequent enzymes in the heme biosynthetic pathway, uroporphyrinogen III synthase and uroporphyrinogen decarboxylase, are deactivated by heating so that their activity does not interfere with the PBGD assay. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is used to monitor the production of uroporphyrinogen I and thus measure the PGBD activity. A simple and efficient workup using liquid-liquid extraction with >90% product recovery was employed to avoid separation by liquid chromatography. The assays show good reproducibility (+/-3.3%) and linear dependence of the uroporphyrinogen I formation on incubation time and protein amount. The Km of PGBD for Porphobilinogen was measured as 11.2 +/- 0.5 microM with Vmax of 0.0041 +/- 0.0002 microM/(min.mg of hemoglobin). The coefficient of variation of PBGD activity among several unaffected individuals (12%) is significantly lower than the decrease due to acute intermittent porphyria (50%).
Leif A. Eriksson - One of the best experts on this subject based on the ideXlab platform.
-
catalytic mechanism of Porphobilinogen synthase the chemical step revisited by qm mm calculations
Journal of Physical Chemistry B, 2012Co-Authors: Boxue Tian, Edvin Erdtman, Leif A. ErikssonAbstract:Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give Porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C–C bond formation first mechanism and the C–N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism.
-
catalytic mechanism of Porphobilinogen synthase the chemical step revisited by qm mm calculations b
The Journal of Physical Chemistry, 2012Co-Authors: Boxue Tian, Edvin Erdtman, Leif A. ErikssonAbstract:Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give Porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C–C bond formation first mechanism and the C–N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism.
-
computational insights into the mechanism of Porphobilinogen synthase
Journal of Physical Chemistry B, 2010Co-Authors: Edvin Erdtman, Eric A C Bushnell, James W Gauld, Leif A. ErikssonAbstract:Porphobilinogen synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of Porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersub ...