Alpha ketoglutarate

Abstract: FeII/alpha-ketoglutarate (alphaKG)-dependent hydroxylases catalyze an amazing diversity of reactions that result in protein side-chain modifications, repair of alkylated DNA/RNA, biosynthesis of antibiotics and plant products, metabolism related to lipids, and biodegradation of a variety of compounds. These enzymes possess a beta-strand "jellyroll" structural fold that contains three metal-binding ligands found in a His1-X-Asp/Glu-Xn-His2 motif. The cosubstrate, alphaKG, chelates FeII using its C-2 keto group (binding opposite the Asp/Glu residue) and C-1 carboxylate (coordinating opposite either His1 or His2). Oxidative decomposition of alphaKG forms CO2 plus succinate and leads to the generation of an FeIV-oxo or other activated oxygen species that hydroxylate the primary substrate. The reactive oxygen species displays alternate reactivity in related enzymes that catalyze desaturations, ring expansions, or ring closures. Other enzymes resemble the FeII/alphaKG-dependent hydroxylases in terms of protein structure or chemical mechanism but do not utilize alphaKG as a substrate. This review describes the reactions catalyzed by this superfamily of enzymes, highlights key active site features revealed by structural studies, and summarizes results from spectroscopic and other approaches that provide insights into the chemical mechanisms.

Abstract: Our attention was drawn to the possibility of studying enzyme induction in tissue culture by a brief report from Pitot et al.1 that the Reuber hepatoma in tissue culture showed an increase in tyrosine transaminase activity in response to treatment with hydrocortisone.' Through the courtesy of Dr. H. P. Morris of the National Cancer Institute, we obtained primary cultures from rats containing two lines of hepatomas in the ascites form. Each of these resulted in a permanent tissue culture line. In this paper we describe the characteristics of one of these lines in which glucocorticoids induce a rapid, substantial increase in the activity of tyrosine a-ketoglutarate transaminase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5). While being carried in serial transfer for over a year, this cell line (designated HTC for hepatoma tissue culture) has remained stable as to growth and inducibility. Furthermore, from single cells of the original line, clones have been isolated which continue to show the same characteristics. Materials and Methods.-Sera were obtained from Microbiological Associates; the NIH medie unit prepared Swim's medium. Merck, Inc., very kindly supplied the dexamethasone phosphata (Dx). Porcine kidney p-hydroxyphenyl pyruvate keto-enol tautomerase (4.3 K units/ml) was purchased from Sigma Chemical Co., centrifuged at 20,000 g for 10 min, the pellet discarded, and the soluble fraction stored frozen. The cell line described here came from an ascites tumor2 which in turn had been derived from a solid hepatoma (#7288c) originally induced by feeding male Buffalo rats a diet containing 0.04% N,N '-2,7-fluorenylenebis-2,2,2-trifluoroacetamide for 12.4 months.3 4 Primary culture was carried out by sterile peritoneal puncture and withdrawal of 0.1 ml ascitic fluid which was placed in a T30 culture flask to which 5 ml growth medium was at once added. After an initial lag of a few days, a layer of epithelioid cells grew out. For the first 8 months, growth was maintained in tightly stoppered bottles in a standard laboratory incubator, but since then a humidified CO2 incubator running with 3% CO2-97% air has been used with the bottles stoppered loosely. The growth medium was Swim's medium 77 (S77). S77 has the same composition as S103 described by Swim and Barker,5 except that hydroxyproline was omitted, the serine concentration was 0.2 mM, and choline bitartrate was substituted for choline chloride. S77, when used to support growth, was supplemented with 20% bovine serum and 5% fetal bovine serum. Penicillin G, 106 units/ml, streptomycin sulfate, 12.5 ,g/ml, were added for routine culturing; however, periodically they have been omitted for several days and the medium was then cultured for bacterial contamination. Checks for PPLO contamination also have been carried out by plating on \"mycoplasma agar\" as described by Hayflick.6 No mycoplasmas have been found. Periodically, cells have been frozen in 5% glycerol-95% growth medium by standard techniques7 and stored in liquid nitrogen. Upon thawing after as much as a year of such storage, HTC cells exhibited the same growth and induction as the original line. Chromosome preparations, stained with GCiemsa, were carried out by a slight modification of the method of Tjio and Puck.8 For enzyme induction studies, growth medium was replaced by serum-free S77; then the cells were gently shaken free, pooled, and apportioned as needed for a given experiment. Tyrosine transaminase was assayed as follows: Each aliquot of cells was centrifuged from the induction medium at 600 g for 5 min at 00, twice washed and recentrifuged with aliquots of 0.15 AI sodium phosphate, p11 7.9 at 0)-4, and the resulting pellet frozen. To the frozen pellet 0.5 il

Abstract: Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH- or FH-deficient cells, HIF activation is due to HIF1α stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIFα prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, α-ketoglutarate. We designed and synthesized cell-permeating α-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate- or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular α-ketoglutarate. Introduction of α-ketoglutarate derivatives restores normal PHD activity and HIF1α levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction.

Abstract: The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-l-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme l-Thr-S-SyrB1. SyrB2 is a member of the nonheme FeII α-ketoglutarate-dependent enzyme superfamily, and requires O2 and α-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of l-Thr-S-SyrB1. Chlorination of l-Thr-S-SyrB1 was validated by thioesterase-mediated release of l-Thr and 4-Cl-l-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with l-[14C]Thr and [36Cl-] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme FeII enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.