Condensation Of Glycine Research Articles

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis

The first enzyme of haem biosynthesis, ALAS (5-aminolaevulinic acid synthase), catalyses the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolaevulinic acid, CO(2) and CoA. The crystal structure of Rhodobacter capsulatus ALAS provides the first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and structural protein rearrangements, multiple ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-oxoadipate intermediate. Further evidence was obtained by the pH-dependent formation of quinonoid II from the product 5-aminolaevulinic acid. The function of Arg(21), Thr(83), Asn(85) and Ile(86), all involved in the co-ordination of the succinyl-CoA substrate carboxy group, were analysed kinetically. Arg(21), Thr(83)and Ile(86), all of which are located in the second subunit to the intersubunit active site, were found to be essential. Their location in the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization of L-alanine by the ALAS variant T83S indicated the importance of this residue for the selectiveness of binding with the glycine substrate compared with related amino acids. Asn(85) was found to be solely important for succinyl-CoA substrate recognition and selectiveness of binding. The results of the present study provide a novel dynamic view on the structural basis of ALAS substrate-binding and catalysis.

A comparative study of the catalysis of peptide bond formation by oxide surfaces

It is well-known that amino acids deposited on some inorganic oxides undergo peptidic condensation. It is seldom realised however that a large diversity of behaviours can be observed in such systems. Here we use the apparently simple case of glycine-non-porous silica as a reference system, in which glycine (Gly) dimerisation to diketopiperazine (DKP) is easy to evidence, especially when using TG in combination with NMR. We then proceed to compare it with other AA deposited on the same support on the one hand, with Gly deposited on other mineral surfaces on the other hand. In a final section, we provide more detailed mechanistic information on the glycine condensation process on silica, including kinetic data and a (13)C solid-state NMR follow up of the species at various stages of thermal condensation. The best mechanism to rationalise these data involves a crucial step of isomerisation from zwitterion to neutral glycine, and the participation of several distinct types of surface sites probably consisting of silanol ensembles.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Thiamin biosynthesis: still yielding fascinating biological chemistry

The present paper describes the biosynthesis of the thiamin thiazole in Bacillus subtilis and Saccharomyces cerevisiae. The two pathways are quite different: in B. subtilis, the thiazole is formed by an oxidative condensation of glycine, deoxy-D-xylulose 5-phosphate and a protein thiocarboxylate, whereas, in S. cerevisiae, the thiazole is assembled from glycine, NAD and Cys205 of the thiazole synthase.

1-Hydroxy-8-methyl-1,4,8-triazaspiro[4.5]decan-2-one and its (±)-3-methyl homologue: regioselective synthesis and in vivo evaluation as adjuvants in leukemia chemotherapy

Condensation of glycine and dl-alanine hydroxamic acids with 1-methyl-4-piperidone regioselectively gave the corresponding titled spiro piperidine hydroxyimidazolidinones capable of enhancing sensitivity of P388 and L1210 leukemias in mice models to the known anticancer drugs cisplatin and cyclophosphamide.

Serine 254 Enhances an Induced Fit Mechanism in Murine 5-Aminolevulinate Synthase

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 A of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K(m)(SCoA) and k(cat) values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased k(cat) without altering K(m)(SCoA). Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved approximately 3-fold, whereas that of S254T diminished approximately 3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire alpha-oxoamine synthase family of PLP-dependent enzymes.

Trafficking of heme and porphyrins in metazoa.

Trafficking of heme and porphyrins in metazoa.

Formation versus hydrolysis of the peptide bond from a quantum-mechanical viewpoint: The role of mineral surfaces and implications for the origin of life.

The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era.

Synthesis of β-hydroxy-α-amino acids with a reengineered alanine racemase

The Y265A mutant of alanine racemase (alrY265A) was evaluated as a catalyst for the synthesis of β-hydroxy-α-amino acids. It promotes the PLP-dependent aldol condensation of glycine with a range of aromatic aldehydes. The desired products were obtained with complete stereocontrol at C α (ee > 99%, D) and moderate to high selectivity at C β (up to 97% de). The designed enzyme is thus similar to natural d-threonine aldolases in its substrate specificity and stereoselectivity. Moreover, its ability to utilize alanine as an alternative donor suggests an expanded scope of potential utility for the production of biologically active compounds.

Transient Kinetic Studies Support Refinements to the Chemical and Kinetic Mechanisms of Aminolevulinate Synthase

5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.

Adsorption and Thermal Condensation of Glycine on Kaolinite

Glycine was adsorbed on the surface of kaolinite from aqueous solution with variable concentrations and pH values. The TG/DTA, FT-IR and in situ DRIFT techniques were employed to characterize the adsorption and thermal condensation performances of glycine on kaolinite. The results of the adsorption experiment indicated that the weakly acidic solution favored the adsorption greatly, and gave the largest adsorption amount, however, the adsorption isotherm did not meet the Langmuir model. FT-IR results showed that in the strongly acidic, weakly acidic, and basic solutions, the glycine exists in the form of cation, zwitterion, and anion, respectively. In the weakly acidic solution, the hydrogen bond interaction between -NH+3 and ≡S-O- groups should be the driving force of the adsorption, the hydroxylation of ≡S-O- group in strongly acidic solution and the conversion of -NH+3 to -NH2 in the basic solution were the main reasons for the decrease of adsorption amount. The in situ DRIFT results revealed that linear dimer of glycine was formed during 110-160 ℃ region. Up to 210 ℃, the dimer underwent further intra-molecular condensation to form the cyclodimer diketopiperazine (DKP). During the reaction, no any evidence of the formation of R-CO-O-S(S=Si or Al) intermediates from the esterification reaction was found. The reaction should proceed by the self-condensation mechanism promoted by the hydrogen bond interaction between glycine and kaolinite. The presence of the kaolinite has remarkably decreased the temperature of the condensation reaction.

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

PGC-1α: Looking behind the Sweet Treat for Porphyria

Porphyrias are a group of genetic disorders caused by mutations in enzymes of the heme biosynthesis pathway. Acute attacks of porphyria, reputedly the disease that incapacitated the British sovereign King George III (see this Cell cover), are precipitated by fasting and are treated by infusing heme or glucose, although the underlying molecular mechanisms remain unclear. In this issue of Cell, reveal that a transcriptional coactivator called PGC-1alpha is a key player in both induction of porphyria by fasting and amelioration of the symptoms by glucose treatment.

δ‐Aminolevulinic acid biosynthesis in Ustilago maydis

AbstractA biosynthetic precursor of tetrapyrroles, δ‐aminolevulinic acid (ALA), can be formed via two pathways: enzymatic condensation of glycine and succinyl‐CoA by ALA synthase in animal mitochondria and some fungi, and the C5 pathway converting glutamate to ALA in plants, algae, archaea, and most bacteria. The two pathways are distinguishable using specifically radiolabeled compounds. The C1 of glutamate is lost during conversion to succinate in the TCA cycle, and the C2 of glycine is lost during conversion to acetyl‐CoA on the way to glutamate. Desalted high‐speed supernatants of Ustilago maydis sporidia extracts were assayed using specifically radiolabeled substrates. A significant amount of radiolabel was incorporated into ALA from 2‐[14C]glycine. No radiolabel was incorporated into ALA from 1‐[14C]glutamate. These results indicate that the basidiomycete yeast, Ustilago maydis, has active ALA synthase. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Enantioselective Synthesis of the Carbacephem Antibiotic Loracarbef via Mitsunobu and Dieckmann Cyclization from an Unnatural Amino Acid.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Enantioselective synthesis of the carbacephem antibiotic loracarbef via Mitsunobu and Dieckmann cyclization from an unnatural amino acid

The nucleus of the carbacephem antibiotic loracarbef was synthesized in a highly efficient and enantioselective fashion from 2 S,3 S-2-amino-3-hydroxy-6-heptenoic acid (AHHA), which was derived from enzyme-catalyzed condensation of glycine and 4-pentenaldehyde. The bicyclic framework of this compound was established through sequential Mitsunobu reaction and aldol condensations.

Catalytic and Selective Conversion of Glycine into Serine by the Reaction with Formaldehyde in a Neutral Aqueous Solution.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Copper complexes as chemical nucleases

Redox active mononuclear and binuclear copper(II) complexes have been prepared and structurally characterized. The complexes have planar N-donor heterocyclic bases like 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) and dipyridophenazine (dppz) ligands that are suitable for intercalation to B-DNA. Complexes studied for nuclease activity have the formulations [Cu(dpq)2(H2O)] (ClO4)2.H2O (1), [CuL(H2O)2(μ-ox)](ClO4)2 (L = bpy,2; phen,3; dpq,4; and dppz,5) and [Cu(L)(salgly)] (L = bpy,6; phen,7; dpq,8; and dppz,9), where salgly is a tridentate Schiff base obtained from the condensation of glycine and salicylaldehyde. The dpq complexes are efficient DNA binding and cleavage active species. The dppz complexes show good binding ability but poor nuclease activity. The cleavage activity of thebis-dpq complex is significantly higher than thebis-phen complex of copper(II). The nuclease activity is found to be dependent on the intercalating nature of the complex and on the redox potential of the copper(II)/copper(I) couple. The ancillary ligand plays a significant role in binding and cleavage activity.

Catalytic and Selective Conversion of Glycine into Serine by the Reaction with Formaldehyde in a Neutral Aqueous Solution

Abstract Di- or trinuclear Cu(II) complexes efficiently catalyzed the condensation of glycine with formaldehyde to yield serine at pH 7.3, 50 °C.