487-90-1Relevant academic research and scientific papers
Structure of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin
Heinemann, Ilka U.,Schulz, Claudia,Schubert, Wolf-Dieter,Heinz, Dirk W.,Wang, Yang-G.,Kobayashi, Yuichi,Awa, Yuuki,Wachi, Masaaki,Jahn, Dieter,Jahn, Martina
, p. 267 - 272 (2010)
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
Direct assay of δ-Aminolevulinic acid dehydratase in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry
Choiniere, John R.,Scott, C. Ronald,Gelb, Michael H.,Turecek, Frantisek
, p. 6730 - 6736 (2010)
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.
Synthesis of porphobilinogen via a novel ozonide cleavage reaction
Jacobi,Li
, p. 9307 - 9312 (2001)
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.
A CONVENIENT AND VERSATILE SYNTHESIS OF PORPHOBILINOGEN
Adamczyk, Maciej,Reddy, Rajarathnam E.
, p. 9121 - 9124 (1995)
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.
Deuterium isotope effects on porphobilinogen synthesis catalysed by 5-aminolaevulinic acid dehydratase
Appleton, Diana,Leeper, Finian J.
, p. 1191 - 1194 (1996)
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
Handling heme: The mechanisms underlying the movement of heme within and between cells
Donegan, Rebecca K.,Moore, Courtney M.,Hanna, David A.,Reddi, Amit R.
, p. 88 - 100 (2018/08/21)
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.
Pseudomonas aeruginosa porphobilinogen synthase assembly state regulators: Hit discovery and initial SAR studies
Reitz, Allen B.,Ramirez, Ursula D.,Stith, Linda,Du, Yanming,Smith, Garry R.,Jaffe, Eileen K.
, p. 175 - 188 (2012/11/07)
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.
Synthesis of [3-13C]-, [4-13C]- and [11- 13C]-porphobilinogen
Dawadi, Prativa B. S.,Schulten, Els A. M.,Lugtenburg, Johan
experimental part, p. 341 - 349 (2011/07/08)
[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
Probing the active site of rat porphobilinogen synthase using newly developed inhibitors
Li, Nan,Chu, Xiusheng,Liu, Xiaojun,Li, Ding
body text, p. 33 - 40 (2009/05/30)
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.
Stereochemistry and mechanism of the conversion of 5-aminolaevulinic acid into porphobilinogen catalysed by porphobilinogen synthase
Goodwin, Catherine E.,Leeper, Finian J.
, p. 1443 - 1446 (2007/10/03)
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.
