Isolation, purification and properties of an intermediate in 3-deoxy-D-manno-octulosonic acid--lipid A biosynthesis.

Incomplete lipid A has been purified from a mutant of Salmonella typhimurium which is temperature-sensitive both in synthesis of 3-deoxy-D-manno-octulosonic acid 8-phosphate (dOclA-8-P) and in growth. Pulse-chase experiments have shown that the incomplete lipid A molecule is the intermediate in the biosynthesis of the dOclA-lipid A portion of lipopolysaccharides. The purification procedure included DEAE-cellulose chromatography and electrodialysis. A highly water-soluble precursor material was obtained, consisting of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio of 1:1.2:2.1. Labeling experiments as well as chemical degradation procedures revealed the precursor molecule to be composed of a diphosphorylated glucosamine-disaccharide carrying two amide-linked and two ester-linked 3-hydroxymyristic acids. In contrast to the complete dOclA-lipid A part, the intermediate lacks 3-deoxy-D-manno-octulosonic acid as well as nonhydroxylated fatty acids. On the basis of these findings a pathway for the final steps in dOclA-lipid A biosynthesis is proposed.     

Evidence implicating dimethylsulfoniopropionaldehyde as an intermediate in dimethylsulfoniopropionate biosynthesis.

3-Dimethylsulfoniopropionate (DMSP) is an osmoprotectant accumulated by certain flowering plants and algae. In Wollastonia biflora (L.) DC. (Compositae) the first intermediate in DMSP biosynthesis has been shown to be S-methylmethionine (SMM) (A.D. Hanson, J. Rivoal, L. Paquet, D.A. Gage [1994] Plant Physiol 105: 103-110). Other possible intermediates were investigated by radiolabeling methods using W. biflora leaf discs. In pulse-chase experiments with [35S]SMM, 3-dimethylsulfoniopropionaldehyde (DMSP-ald) acquired label rapidly and lost it during the chase period. Conversely, 3-dimethylsulfoniopropylamine (DMSP-amine), 3-dimethylsulfoniopropionamide (DMSP-amide), and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB) labeled slowly and continuously during both pulse and chase. When unlabeled compounds were supplied along with [35S]SMM, DMSP-ald promoted [35S]DMSP-ald accumulation but DMSHB, DMSP-amide, and DMSP-amine had no such trapping effect. These data indicate that DMSP-ald is an intermediate in DMSP biosynthesis and that the other three compounds are not. Consistent with this, [35S]DMSHB was not metabolized to DMSP. Although [14C]DMSP-amine and [14C]DMSP-amide were converted slowly to DMSP, similar or higher conversion rates were found in plants that do not naturally accumulate DMSP, indicating that nonspecific reactions were responsible. These nonaccumulating species did not form [35S]DMSP-ald from [35S]SMM, implying that DMSP-ald is specific to DMSP biosynthesis. W. biflora leaf discs catabolized supplied sulfonium compounds to dimethylsulfide at differing rates, in the order DMSP-ald >> DMSP-amine > SMM > DMSP-amide > DMSHB > DMSP.     

Tetrahydro-sepiapterin is an intermediate in tetrahydrobiopterin biosynthesis

7,8-Dihydrobiopterin is not an intermediate in the de novo biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations. However, N-acetyl-serotonin inhibition of sepiapterin reductase, an enzyme whose previously only known function was the reduction of sepiapterin to 7,8-dihydrobiopterin, completely inhibited biosynthesis of tetrahydrobiopterin by bovine adrenal medulla extracts. We have now shown that sepiapterin reductase catalyzes the reduction of tetrahydro-sepiapterin to tetrahydrobiopterin and that this reaction is N-acetyl-serotonin-sensitive. A new pathway for tetrahydrobiopterin biosynthesis is proposed which takes these observations into account and which involves tetrahydro intermediates.     

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.     

The late steps of anaerobic heme biosynthesis in E. coli: role for quinones in protoporphyrinogen oxidation.

The anaerobic oxidation of protoporphyrinogen with fumarate as electron acceptor in cell-free extracts of $\text{E. coli}$ is inhibited by ultra-violet irradiation. The activity of irradiated extracts is restored by addition of menadione and the restored activity is blocked by the electron-transport inhibitor 2-heptyl-4-hydroxy quinoline-N-oxide. These observations suggest that quinones are required as electron transport carriers at this late step in the pathway of anaerobic heme biosynthesis. These findings have important implications both for the mechanism of anaerobic heme synthesis and for the physiology of cytochrome biosynthesis in anaerobic microorganisms.     

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.     

Role of Ngamma-acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine.

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Ngamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A (1)H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Enzymes of heme biosynthesis

 Heme is a necessary component in a variety of oxygen-binding proteins and electron-transfer proteins, and as such it occupies a central role in cellular and organismal metabolism. With only rare exceptions, organisms that utilize heme possess the entire biosynthetic pathway to produce this tetrapyrrole compound. The enzymes involved catalyze a variety of interesting reactions and utilize both common and unique cofactors and metals. Aminolevulinate dehydratase from all organisms and ferrochelatase from higher animals are both metalloenzymes, while 5-aminolevulinate synthase contains pyridoxal phosphate, and porphobilinogen deaminase possesses a unique dipyrrole cofactor. Two pathway enzymes catalyze multiple decarboxylations and yet have no cofactors, and one enzyme catalyzes a six-electron oxidation with a single FAD. To add additional scientific interest there exist biochemically and clinically distinct human genetic diseases for every step in this pathway. Received: 12 March 1997 / Accepted: 8 May 1997     

Hydroxyurea,a natural metabolite and an intermediate in d-cycloserine biosynthesis in streptomyces garyphalus

It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [¹⁴C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-¹⁴C]-arginine and l-[ureido-¹⁴C]-citrulline. The ¹⁵N of H₂NCO¹⁵NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the ¹⁵N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [¹⁵N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine.     

Isoporphyrin intermediate in heme oxygenase catalysis. Oxidation of alpha-meso-phenylheme

Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the alpha-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin pi-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of alpha-meso-phenylheme-IX, alpha-meso-(p-methylphenyl)-mesoheme-III, and alpha-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593-42604), only the alpha-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced alpha-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation.