- Structure of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin
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The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a Ki of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-A resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed. Copyright
- Heinemann, Ilka U.,Schulz, Claudia,Schubert, Wolf-Dieter,Heinz, Dirk W.,Wang, Yang-G.,Kobayashi, Yuichi,Awa, Yuuki,Wachi, Masaaki,Jahn, Dieter,Jahn, Martina
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- Direct assay of δ-Aminolevulinic acid dehydratase in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry
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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 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95-99%. The assay shows good reproducibility and low background, requires a simple workup, and uses a commercially available substrate.
- Choiniere, John R.,Scott, C. Ronald,Gelb, Michael H.,Turecek, Frantisek
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- Synthesis of porphobilinogen via a novel ozonide cleavage reaction
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Porphobilinogen lactam methyl ester (3a) has been prepared in seven steps, and ～20-30% overall yield, beginning with furfurylamine (4a).24 Hydrolysis of 3a following the literature procedure then gave porphobilinogen (1). A key intermediate in our synthesis of 3a is the 7-oxonorbornene derivative 7a, which was derived from 4a utilizing a tandem Johnson ortho ester Claisen rearrangement followed by intramolecular Diels - Alder cyclization (five steps, 55-65%).24 Interesting steric accelerating effects were observed in this sequence. Conversion of 7a to 3a was then accomplished employing a novel ozonide cleavage/oxidation reaction, which generated tetrahydrofurans 16a, 32, and 33 in the proper oxidation state for direct aminolysis to pyrrole 3a. A mechanism is proposed for the ozonide cleavage/oxidation that accounts for the observed stereoselectivity of this step.
- Jacobi,Li
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- A CONVENIENT AND VERSATILE SYNTHESIS OF PORPHOBILINOGEN
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Porphobilinogen (PBG, 1) was synthesized from 2-cyano-3,4-substituted pyrrole 3, which was obtained by condensation of α-acetoxynitro compound 4b with isocyanoacetonitrile (5), via functional group transformation, in good yield.
- Adamczyk, Maciej,Reddy, Rajarathnam E.
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- Deuterium isotope effects on porphobilinogen synthesis catalysed by 5-aminolaevulinic acid dehydratase
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Deuteriation of 5-aminolaevulinic acid (ALA) at C-5 has no effect on the rate of porphobilinogen synthesis by ALA dehydratase from Bacillus subtilis but deuteriation at C-3 gave isotope effects on kcat and kcat/KM of 3.4 and 2.3 respectively. Reisolated ALA after 50% reaction shows no significant loss of deuterium at C-3, indicating that it is probably the first deprotonation at this carbon which is rate-determining. Copyright
- Appleton, Diana,Leeper, Finian J.
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- Handling heme: The mechanisms underlying the movement of heme within and between cells
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Heme is an essential cofactor and signaling molecule required for virtually all aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely transported and trafficked from the site of synthesis in the mitochondria or uptake at the cell surface, to hemoproteins in most subcellular compartments. While heme synthesis and degradation are relatively well characterized, little is known about how heme is trafficked and transported throughout the cell. Herein, we review eukaryotic heme transport, trafficking, and mobilization, with a focus on factors that regulate bioavailable heme. We also highlight the role of gasotransmitters and small molecules in heme mobilization and bioavailability, and heme trafficking at the host-pathogen interface.
- Donegan, Rebecca K.,Moore, Courtney M.,Hanna, David A.,Reddi, Amit R.
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- Pseudomonas aeruginosa porphobilinogen synthase assembly state regulators: Hit discovery and initial SAR studies
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Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of the essential heme, chlorophyll and vitamin B12 heme pigments. PBGS activity is regulated by assembly state, with certain oligomers exhibiting biological activity and others either partially or completely inactive, affording an innovative means of allosteric drug action. Pseudomonas aeruginosa PBGS is functionally active as an octamer, and inactive as a dimer. We have identified a series of compounds that stabilize the inactive P. aeruginosa dimer by a computational prescreen followed by native PAGE gel mobility shift analysis. From those results, we have prepared related thiadiazoles and evaluated their ability to regulate P. aeruginosa PBGS assembly state. ARKAT USA, Inc.
- Reitz, Allen B.,Ramirez, Ursula D.,Stith, Linda,Du, Yanming,Smith, Garry R.,Jaffe, Eileen K.
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p. 175 - 188
(2012/11/07)
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- Probing the active site of rat porphobilinogen synthase using newly developed inhibitors
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The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Porphobilinogen synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. In the present study, a variety of new substrate analogs and reaction intermediate analogs were synthesized, which were used as probes for studying the active site of rat porphobilinogen synthase. The compounds 1, 3, 6, 9, 14, 16, and 28 were found to be competitive inhibitors of rat porphobilinogen synthase with inhibition constants ranging from 0.96 to 73.04 mM. Compounds 7, 10, 12, 13, 15, 17, 18, and 26 were found to be irreversible enzyme inhibitors. For irreversible inhibitors, loose-binding inhibitors were found to give stronger inactivation. The amino group and carboxyl group of the analogs were found to be important for their binding to the enzyme. This study increased our understanding of the active site of porphobilinogen synthase.
- Li, Nan,Chu, Xiusheng,Liu, Xiaojun,Li, Ding
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body text
p. 33 - 40
(2009/05/30)
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- Synthesis of [3-13C]-, [4-13C]- and [11- 13C]-porphobilinogen
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[4-13C]-porphobilinogen 1a, [3-13C]-porphobilinogen 1b and [11-13C]-porphobilinogen 1c are prepared from [1- 13C]-3-(tetrahydropyran-20-yloxy)-propionaldehyde 2a, methyl [4- 13C]-4-nitrobutyrate 3b and [1-13C]-isocyanoacetonitrile 5c, respectively. The building blocks 2, 3 and 5 can be prepared efficiently in any isotopomeric form. Via base-catalyzed condensation of these building blocks porphobilinogen can be enriched with 13C and 15N stable isotopes at any position and combination of positions. Copyright
- Dawadi, Prativa B. S.,Schulten, Els A. M.,Lugtenburg, Johan
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experimental part
p. 341 - 349
(2011/07/08)
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- Stereochemistry and mechanism of the conversion of 5-aminolaevulinic acid into porphobilinogen catalysed by porphobilinogen synthase
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Several (3R)- and (3S)-Deuterated forms of 5-aminolaevulinic acid were synthesized. The resultant (3R)-form shows a significantly larger isotope effect when incubated with porphobilinogen synthase from bovine liver and from Bacillus subtilis. Based on the results of this study and on available crystal structures, a modified mechanism for the enzyme reaction was established.
- Goodwin, Catherine E.,Leeper, Finian J.
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p. 1443 - 1446
(2007/10/03)
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- Preparation of 2-carboxy-3,4-substituted pyrrole haptens and synthesis of porphobilinogen
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2-Carboxy-3,4-substituted pyrrole 1, the porphobilinogen (PBG, 2) like hapten was prepared via condensation of α-acetoxynitro compound (3) with benzyl isocyanoacetate (4) and further functional group transformations in good yields. Also, the hapten 1 was converted to PBG (2).
- Adamczyk, Maciej,Reddy, Rajarathnam E.
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p. 2325 - 2326
(2007/10/03)
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- A new methodology for the preparation of 2-cyanopyrroles and synthesis of porphobilinogen
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Condensation of α-acetoxynitro compounds 4a-f with isocyanoacetonitrile (5) using DBU in THF afforded 2-cyano-3,4-substituted pyrroles 6a-f, in good yield. Porphobilinogen (PBG, 12), the key building block for the preparation of tetrapyrrolic natural products, was synthesized from 2-cyano-3,4-substituted pyrrole 6f, in four steps.
- Adamczyk, Maciej,Reddy, Rajarathnam E.
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p. 14689 - 14700
(2007/10/03)
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- The nonenzymatic cyclic dimerisation of 5-aminolevulinic acid
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The nonenzymatic cyclic dimerisation of 5-aminolevulinic aid (5-ALA) leads to the formation of a pyrazine (3) and, under some circumstances, pseudo-porphobilinogen (1). On the other hand, the enzyme-catalysed process leads to porphobilinogen (PBG). The products of the former reaction were identified from their NMR spectra and mechanisms for their formation are proposed.
- Butler,George
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p. 7879 - 7886
(2007/10/02)
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- Mechanism and Stereochemistry of the Porphobilinogen Deaminase and Protoporphyrinogen IX Oxidase Reactions: Stereospecific Manipulation of Hydrogen Atoms at the Four Methylene Bridges during the Biosynthesis of Haem
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Porphobilinogen (PBG) with an enantiomeric excess greater than 81percent was prepared in a coupled enzyme system from glycine.Incorporation of this chiral PBG into haem using a broken cell enzyme system from chicken blood showed retention of only one tritium per haem molecule.Degradation of the haem showed that tritium had been incorporated specifically into the C-10 position of haem.Control experiments with -PBG showed an equal distribution of tritium at all four meso positions of haem.The implications of this result are discussed in terms of the mechanism of two enzymes in haem biosynthesis: porphobilinogen deaminase (hydroxymethylbilane synthase) and protoporphyrinogen IX oxidase.
- Jones, Christopher,Jordan, Peter M.,Akhtar, Muhammad
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p. 2625 - 2633
(2007/10/02)
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- Pyrrole chemistry. XXVI. A synthesis of porphobilinogen from pyrrole
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A synthetic strategy has been developed which enables pyrrole to be used as the starting material for the synthesis of 2,3,4-trisubstituted pyrroles of the type used to construct porphyrins.The total synthesis of porphilinogen has been accomplished as a demostration of the applicability of the approach.
- Demopoulos, Basil J.,Anderson, Hugh J.,Loader, Charles E.,Faber, Kurt
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p. 2415 - 2422
(2007/10/02)
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- CONDENSATION PRODUCTS OF THE PORPHYRIN PRECURSOR 5-AMINOLEVULINIC ACID
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Condensation of the biogenetic porphyrin precursor 5-aminolevulinic acid (1) in alkaline solution yields besides some porphobilinogen (2) a dihydropyrazine (6) as the predominant product, which was isolated and characterized after dehydrogenation to the stable pyrazine (7a).This ends a longstanding uncertainty and reveals that the azomethine reaction, as can be expected for α-aminoketones, is strongly preferred by 5-aminolevulinic acid (1) under nonenzymatic conditions.A nor-porphobilinogen (9) was formed by condensation of a protected aminoacetoacetic ester with (1).
- Franck, Burchard,Stratmann, Helmut
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p. 919 - 923
(2007/10/02)
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