- Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase
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5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5′-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the kcat values of the variants differed significantly (4.4 ± 0.2 vs. 21.6 ± 0.7 min-1) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.
- Turbeville, Tracy D.,Zhang, Junshun,Christopher Adams,Hunter, Gregory A.,Ferreira, Gloria C.
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- Light triggering of 5-aminolevulinic acid from fused coumarin ester cages
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The evaluation of the photorelease of 5-aminolevulinic acid (5-ALA), a small molecule which has considerable interest in the area of medicine as a photosensitizer prodrug in PDT and cosmetic treatments, and in agriculture as a herbicide/insecticide, was carried out by using a series of fused coumarin derivatives with different substituents and ring fusions in the preparation of 5-ALA photosensitive ester cages, in order to tune the photophysical and photolytic properties of the resulting conjugates. This study was intended to assess the viability of the photorelease of 5-ALA from lipophilic conjugates since it has hydrophilic character, does not penetrate efficiently through the skin or cell membranes and appropriate derivatisation can enhance its lipophilicity and its application scope in biological environment. Photolytic studies were performed on the ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80 : 20 solutions as solvent. The data obtained confirmed the suitability of the tested photosensitive moieties for the release of 5-aminolevulinic acid at the various wavelengths of irradiation. As well as the photolysis, the photophysics of the compounds was characterised by both steady state and time-resolved methods which uncovered the presence of different fluorescing species. Additionally, an on-off experiment was carried out by using two excitation sources (cleave and probe) to enable fluorescence to follow the kinetics of the process and to ascertain optical control over the bond scission.
- Soares, Ana M. S.,Hungerford, Graham,Gon?alves, M. Sameiro T.,Costa, Susana P. G.
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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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- Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase
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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 Ae 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 KmSCoA and kcat values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased kcat without altering Km SCoA. Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ～3-fold, whereas that of S254T diminished ～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 α-oxoamine synthase family of PLP-dependent enzymes.
- Lendrihas, Thomas,Hunter, Gregory A.,Ferreira, Gloria C.
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- Influence of precursors and inhibitor on the production of extracellular 5-aminolevulinic acid and biomass by rhodopseudomonas palustris kG31
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5-Aminolevulinic acid (ALA) and the biomass of photosynthetic bacteria, Rhodopseudomonas palustris KG31, have very high potential for development and exploitation as bioherbicide and biofertilizer respectively. In this work, the effects of two precursors
- Saikeur, Angkana,Choorit, Wanna,Prasertsan, Poonsuk,Kantachote, Duangporn,Sasaki, Ken
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- Process for preparing 5-aminolevulinic acid from 5-chloromethyl furfural
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The invention discloses a process for preparing 5-aminolevulinic acid from 5-chloromethylfurfural. The biomass-based 5-chloromethylfurfural and cheap and easily available potassium phthalimide are used as reaction raw materials to react to obtain ammoniated product 5-(phthalimide) methylfurfural with a yield of 97.1%, and then the 5-ALA is obtained through synthesis steps of photooxidation, reduction, hydrolysis and the like. The structure of the 5-ALA is determined by nuclear magnetic resonance. The purity of the 5-ALA is 96.1% and the total reaction yield is 23.7%. Compared with the traditional chemical synthesis route of the 5-aminolevulinic acid, the process has the advantages of cheap and easily available raw materials, simple and convenient operation, mild reaction conditions, no toxicity and the like, and has a good industrial popularization prospect.
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Paragraph 0025; 0033-0036; 0043-0045; 0052-0054; 0061-0062
(2019/10/29)
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- Catalyst process-based aminolevulinic acid preparation method
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The invention relates to the field of organic chemistry preparation, and discloses a catalyst process-based aminolevulinic acid preparation method. The method comprises the following steps: 1, synthesizing aminolaevulic acid (ALA): reacting a substance represented by molecular formula I with a substance represented by molecular formula II under the catalysis action of active carbon to generate the ALA; 2, filtering: cooling the product obtained in step 1, and filtering the cooled product; 3, forming salt and removing impurities: adding hydrochloric acid to a filtrate obtained in step 2, uniformly mixing hydrochloric acid and the filtrate, centrifuging the obtained solution, and carrying out evaporative concentration on the above obtained supernatant; and 4, purifying: adding a concentrate obtained in step 3 to acetone, fully mixing the concentrate and acetone, and drying the obtained mixture to obtain aminolevulinic acid. The preparation method has the advantages of simple technology, low cost, short production period, energy saving, environmental protection, and achieving of high yield and high purity of the target product.
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Paragraph 0038; 0039; 0040; 0041; 0042; 0043; 0044-0049
(2017/02/09)
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- Study of the stability of the 5-aminolevulinic acid tyrosine ester in aqueous solution
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Photodynamic therapy based on photoactivable porphyrins (PAPs) can treat various dermatological conditions. The side-effects as well as the non-selective or insufficient accumulation of PAPs in the targeted tissues limit performances. We studied the stability in solution at different temperatures (21 °C; 4 °C), different pH values (7.5; 2.0), and as a function of time of 5-aminolevulinic acid's Tyrosine-ester, a molecule presenting interesting properties to selectively produce PAPs in blood vessels after topical application. Solutions of this precursor can be kept up to 24 h at refrigerated temperatures and under acidic pH. At room temperature or physiological pH, they must be prepared minutes before their use. ARKAT-USA, Inc.
- Gay, Sandrine,Martoccia, Carla,Zellweger, Matthieu,Wang, Qian,Wagnieres, Georges
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p. 228 - 238
(2014/06/09)
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- A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis
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A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C5N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C5N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C5N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of ～10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C5N. C 5N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C56 polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C 5N product was observed. This three enzyme pathway is likely the general route to the C5N ring system in other natural products, including the antibiotic moenomycin.
- Zhang, Wenjun,Bolla, Megan L.,Kahne, Daniel,Walsh, Christopher T.
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experimental part
p. 6402 - 6411
(2010/07/04)
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- PROCESS FOR PRODUCING 5-AMINOLEVULINIC ACID HYDROCHLORIDE
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A process for producing 5-aminolevulinic acid hydrochloride crystals which comprises: adsorbing the 5-aminolevulinic acid contained in a crude 5-aminolevulinic acid solution onto a cation-exchange resin; subsequently desorbing the acid with an aqueous solution containing ammonium ions while utilizing a change in the electrical conductivity or pH of the solution resulting from the desorption as an index to recovery to thereby obtain an aqueous solution of high-purity 5-aminolevulinic acid; adding chloride ions to the aqueous solution; and mixing an organic solvent therewith.
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Page/Page column 10-11; 1/3-2/3
(2008/06/13)
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- 5-AMINOLEVULINIC ACID PHOSPHATE SALT, PROCESS FOR PRODUCING THE SAME AND USE THEREOF
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A 5-aminolevulinic acid salt which is useful in fields of microorganisms, fermentation, animals, medicaments, plants and the like; a process for producing the same; a medical composition comprising the same; and a plant activator composition comprising the same.
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Page/Page column 7; 8; 19-20; 23-24
(2008/06/13)
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- Antibiotics napsamycins A-D, process for their production and their use as pharmaceuticals
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This invention relates to new antibacterial antibiotics Napsamycins A - D of the formula Napsamycin A :R1 = H, R2 = uracil Napsamycin B :R1 = CH3, R2 = uracil Napsamycin C :R1 = H, R2 = dihydrouracil Napsamycin D :R1 = CH3, R2 = dihydrouracil from a strain of Streptomyces candidus, Y-82,11372 (DSM 5940).
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