Alternative Biosynthetic Pathway Research Articles

Relationships between phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin metabolism in cultured oligodendrocytes.

In most cell types the major pathway of sphingomyelin synthesis is the direct transfer of the phosphocholine head group from phosphatidylcholine to ceramide catalyzed by the enzyme L-acylsphingosine:phosphatidylcholine phosphocholinetransferase (SM synthase; EC 2.7.8.-). Although this pathway has been demonstrated in brain tissue, its quantitative importance has been questioned. An alternative biosynthetic pathway for sphingomyelin synthesis in brain tissue has been proposed, viz., the direct transfer of phosphoethanolamine from phosphatidylethanolamine to ceramide, followed by methylation of the ethanolamine moiety to a choline group. We have evaluated various possible biosynthetic pathways of sphingomyelin synthesis in rat spinal cord oligodendrocytes, the myelin-forming cells of the CNS, by labeling cells in culture with radiolabeled choline, ethanolamine, or serine. Our results indicate that, in oligodendrocytes, most of the phosphocholine for the biosynthesis of sphingomyelin is provided by phosphatidylcholine, which is predominantly derived from de novo synthesis. No evidence was found for the operation of the alternative pathway via ceramide-phosphoethanolamine. Furthermore, our results indicate that a small pool of phosphatidylcholine is provided by methylation of phosphatidylethanolamine, which in turn is formed preferentially by decarboxylation of phosphatidylserine.

An alternative brassinolide biosynthetic pathway via late C-6 oxidation

6-Deoxoteasterone and 6-deoxotyphasterol were identified in cultured cells of Catharanthus roseus. Feeding experiments with deuterium-labelled substrates revealed the conversions of 6-deoxoteasterone to 6-deoxotyphasterol, and of 6-deoxotyphasterol to 6-deoxocastasterone in cultured cells of C. roseus. Furthermore, the conversion of 3-dehydro-6-deoxoteasterone to 6-deoxotyphasterol was also demonstrated in the cells. These results, together with our previously published data, demonstrate an alternative biosynthetic pathway of brassinolide production: 6-deoxoteasterone→3-dehydro-6-deoxoteasterone→6-deoxotyphasterol→6-deoxocastasterone→castasterone→brassinolide. This is termed the late C-6 oxidation pathway, and it is operated in cultured cells of C. roseus in addition to the early C-6 oxidation pathway.

A novel involvement of the PurG and PurI proteins in thiamine synthesis via the alternative pyrimidine biosynthetic (APB) pathway in Salmonella typhimurium.

Thiamine is thought to be synthesized by two alternative pathways, one involving the first four enzymes of the purine pathway and a second that can function independently of the purine pathway. Insertion mutations in purG and purI prevent thiamine synthesis through the alternative pyrimidine biosynthetic (APB) pathway under aerobic but not anaerobic growth conditions. In contrast, point mutations in purG and purI caused one of three distinct phenotypes: Pur- Apb-, Pur- Apb+, or Pur+ Apb-. Analysis of these three mutant classes demonstrated two genetically separable functions for PurG and PurI in thiamine synthesis. In addition to their known enzymatic role in de novo purine synthesis, we propose that PurG and PurI play a novel, possibly nonenzymatic role in the APB pathway. Suppression analysis of Pur- Apb- mutants identified two new genetic loci involved in the APB pathway, apbB and apbD). We show here that mutations in apbB and apbD cause distinct, allele-specific suppression of the thiamine requirement of purG and purI mutants. Our results suggest that PurG and PurI and one or more components of the APB pathway may function as a complex needed for aerobic function of the APB pathway.

Discovery of a new tetrahydrobiopterin-synthesizing enzyme in the lemon mutant of the silkworm Bombyx mori

A new tetrahydrobiopterin-synthesizing enzyme, which is different from sepiapterin reductase (EC 1.1.1.153), was discovered in the integument of the lemon mutant of the silkworm Bombyx mori. This enzyme converted 6-pyruvoyltetrahydropterin to tetrahydrobiopterin, an essential cofactor in the hydroxylation of aromatic amino acids, in the presence of NADPH. The reaction proceeded via 6-lactoyltetrahydropterin and 1′-hydroxy-2′-oxopropyltetrahydropterin as intermediates. The molecular mass of this enzyme was estimated to be 40 kDa. N-Acetylserotonin, a potent inhibitor of sepiapterin reductase, slightly inhibited the enzymatic reaction. In the presence of 0.5 mM N-acetylserotonin, the formation of tetrahydrobiopterin by sepiapterin reductase purified from the normal strain silkworm was completely inhibited. However, the formation of tetrahydrobiopterin by the enzyme purified from the lemon mutant was inhibited by only about 50%. These results suggest an alternative biosynthetic pathway to tetrahydrobiopterin.

Ungew�hnliche Wege und umweltregulierte Gene der bakteriellen H�mbiosynthese

The majority of bacteria, all investigated archaea and plants form the general precursor molecule of all tetrapyrroles 5-aminolevulinic acid by a unique transformation of transfer RNA bound glutamate. Only the alpha-group of the proteobacteria, mammals and yeast synthesize 5-aminolevulinic acid via the well known condensation of succinyl-CoA and glycine. The late steps in tetrapyrrole biosynthesis also contain alternative biosynthetic pathways for the formation and oxidative decarboxylation of coproporphyrinogen III. Unusual enzymatic reactions including the utilization of two substrate molecules as cofactor by the porphobilinogen deaminase and the formation of a spiro intermediate are involved in the formation of uroporphyrinogen III. The biosynthesis of hemes in bacteria is strictly regulated at the formation of 5-aminolevulinic acid and the oxidative decarboxylation of coproporphyrinogen III. The involved heme biosynthetic genes are regulated by the environmental concentrations of oxygen, iron, nitrate, growth phase and intracellular levels of heme. The current knowledge on the various enzymatic reactions and gene regulatory mechanisms is reviewed.

Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide.

Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.

Biosynthesis of brevianamides A and B: in search of the biosynthetic diels-alder construction

A [ 13C]labeled hypothetical intermediate metabolite that was invoked by Birch in the biogenesis of brevianamides A and B has been synthesized and used in a biosynthetic feeding experiment. No incorporation of this substance was evident nor could it be detected in culture extracts of the producing organism Penicillium brevicompactum. In contrast with this, [ 3H]labeled deoxybrevianamide E was shown to be efficiently incorporated in brevianamides A, B, and E, while [ 3H]labeled brevianamide E was not incorporated at all in these substances. Moreover, brevianamide E has been found not to be an artifact, in contrast with an earlier suggestion found in the literature. These results are discussed in terms of an alternative biosynthetic pathway for the brevianamides.

Biosynthesis of cholic acid accelerated by diabetes: its mechanism and effect of vanadate administration

[7 β- 3H]3α,7α-Dihydroxy-5β-cholestane was infused into the femoral vein of bile-fistula rats. The main metabolites in bile were cholic, chenodeoxycholic, and α-muricholic acids. Cholic acid accounted for only 7.5% of the total biliary [ 3H]bile acids in the normal rats, but 51.8% in the diabetic rats. This increased proportion of cholic acid was partially cancelled by treatment of the diabetic rats with insulin (29.8%) or vanadate (28.8%). These results clearly confirm that an alternative biosynthetic pathway of cholic acid via 3α,7α-dihydroxy-5β-cholestane is accelerated by diabetes, and indicate that vanadate has an insulin-like effect on the formation of cholic acid in an insulin-deficient state.

New aspects of submerged fermentation of Claviceps paspali

Submerged fermentation of Claviceps paspali strain MG-6 was studied with regard to the fate of individual alkaloids in the course of fermentation. Based on liquid-chromatographic data the process can be divided into three phases. The production phase (fermentation days 3–12) is characterized by biosynthesis of the basic genuine alkaloid, lysergic acid alpha-hydroxyethylamide (LAH I). In view of its low stability it becomes epimerized on two asymmetric carbon atoms in the fermentation medium, yielding three other epimers (LAH II–IV). The degradation phase (days 13–18) involves cleavage of LAH I–IV resulting in ergine and erginine. In the post-production phase (days 15–30) the alkaloids succumb to biooxidative reactions that yield mostly 8-hydroxy-derivatives of ergine and erginine. Isolation of paspalic acid 10-hydroxyamide signifies the existence of an alternative biosynthetic pathway for production of simple lysergic acid derivatives.

Quinoline alkaloids. Part 28. The biosynthesis of furoquinolines and other hemiterpenoids in Ptelea trifoliata

Feeding experiments with doubly labelled 4-methoxy-3-prenyl-2-quinolones have shown that the biosynthesis of kokusaginine 8 and maculosidine 9 in P. trifoliata also involves a myrtopsine-like derivative. Contrary to our proposed pathways, the biosynthesis of hemiterpenoid olefines such as ptelefoline 15, in the same plant species, does not appear to involve directly platydesminium salt but possibly also a myrtopsine derivative. On the basis of the results obtained, alternative biosynthetic pathways to these alkaloids are discussed.

Production of 21-deoxyaldosterone by rat adrenal tissue in vitro; evidence for an alternative biosynthetic pathway of aldosterone

Quartered rat adrenal glands transformed labelled 21-deoxyaldosterone into aldosterone in vitro. 21-deoxyaldosterone was released from the quartered rat adrenals in vitro in amounts 10 times lower than those of aldosterone and 18-hydroxycorticosterone. The production of all three steroids was qualitatively dependent on the same regulatory elements (electrolytes, ACTH, exogenous precursors, zonal specificity). However, quantitative differences could be observed. The results support a role for 21-deoxyaldosterone as a facultative precursor of aldosterone and indicate that the regulation of its production shows similarities to that of aldosterone.

Sex pheromone biosynthesis in the processionary moth Thaumetopoea pityocampa by delta‐13 desaturation

In vivo treatments of female sex pheromone glands of the processionary moth, Thaumetopoea pityocampa, with mass-labeled fatty acids showed that (Z)-13-hexadecen-11-ynyl acetate, the main sex pheromone component, is biosynthesized from palmitic acid by the combined action of delta-11 and delta-13 desaturases. The involvement of this unusual delta-13 has been proven by application of [16,16,16-2H3] [1,2-13C2]-hexadecanoic acid to the glands with a resultant incorporation of all labeled atoms into the pheromone and each one of the corresponding intermediates. These results seem to exclude alternative biosynthetic pathways, such as chain shortening and elongation combined with delta-11 desaturation. The delta-11 desaturase responsible for the formation of the triple bond in both the 11-hexadecynoyl and (Z)-13-hexadecen-11-ynoyl intermediates is also an unusual enzyme not previously reported in lepidopteran sex pheromone biosynthesis.

Kinetic analysis of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase.

Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (vT) and hydrolysis (vH) of pyridine nucleotides could be monitored simultaneously. (1) The vT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of vT and vH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively. (2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of Vmax/Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows; Km(NMN) = 0.53 mM, Km(nicotinic acid), as acid form = 15 mM, apparent Vmax = 7.8 mumol/min/mg protein, in the presence of 0.2 M nicotinic acid. (3) The ratio of vT/vH was shown to be dependent on both pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2 x 10(4) at the maximal pH. (4) Nicotinamide, one of the most potent inhibitors for the enzyme-catalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhibition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards. (5) The equilibrium constant of the reaction, NMN + 3-acetylpyridine----3-acetylpyridine mononucleotide + nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.

Influence of Inhibitors of Polyamine Biosynthesis on Polyamine Levels and Growth of Plants

Abstract Inhibitors of enzymes involved in polyamine biosynthesis which stop the growth of bacteria, fungi and animal cell systems were analyzed for their potential to interfere with plant cell systems. Several compounds were found to be potent inhibitors of plant enzymes, namely the decarboxylases of ornithine, arginine and S-adenosylmethionine as well as the aminopropyltransferase. Application of enzyme inhibitors (α-difluoromethylornithine. l-aminooxy-3-aminopropane, a-difluoromethylarginine, methylglyoxal bis(guanyl-hydrazone), cyclohexylamine) or combinations of these on whole plant systems resulted neither in a large decrease in polyamine content nor in a significant growth reduction. The lack of response cannot be explained by hindered inhibitor uptake into plants. Inhibition of single enzymes may induce alternative biosynthetic pathways.

Elevated 18-hydroxyprogesterone, 21-deoxyaldosterone and substance KELLY Ml in patients with 21-hydroxylase deficiency

In the alternative biosynthetic pathway of aldosterone, 18-hydroxyprogesterone and 21-deoxyaldosterone have been considered as precursors. 1. “In vitro”, bovine adrenals were able to transform 21-deoxyaldosterone to aldosterone with a 1-5% and 18-hydroxyprogesterone to aldosterone with a 0.2-1 % yield. 2. Substance Kelly Ml was originally detected in the urine of subjects loaded with large (40-200 mg) amounts of exogenous aldosterone. 3. Recently we have isolated identical (GC/MS) material from the urine of subjects not previously given aldosterone. 4. Comparing the structures of these three steroids we have imagined a transformation pathway leading from 18-hydroxyprogesterone to substance Kelly Ml (Figure). As all three compounds lack the 21-hydroxy group, it seemed logical to suspect their elevated excretion in patients with 21-hydroxylase deficiency.

Kinetic simulation of anticancer drug effects on metabolic pathway fluxes: two case studies.

This paper presents a brief review of applications of kinetic simulation of multi-enzyme networks to the study of antimetabolite drugs used as anticancer agents. Kinetic models consist of systems of nonlinear differential equations that describe changes in concentrations of cellular metabolites with respect to time. Such models have been used to predict the effect of changes in activity of enzymes, or changes in enzyme kinetic parameters, on sensitivity to inhibition. Kinetic simulation has provided insight into several aspects of the biochemical pharmacology of antimetabolites, including drug sensitivity and resistance, and drug-drug interactions. Two specific studies are described in detail. The first concerns the importance of the ratio of competing enzymes in determining the selectivity of inhibitors of one of the competing enzymes, studied by a simple model. The second case study examines the effect of alternative biosynthetic pathways, thede novo and salvage pathways of pyrimidine nucleotide biosynthesis, on the selectivity of antipyrimidine drugs, as studied by a detailed model of 27 reactions of pyrimidine metabolism.

Kinetic simulation of anticancer drug effects on metabolic pathway fluxes: Two case studies

This paper presents a brief review of applications of kinetic simulation of multi-enzyme networks to the study of antimetabolite drugs used as anticancer agents. Kinetic models consist of systems of nonlinear differential equations that describe changes in concentrations of cellular metabolites with respect to time. Such models have been used to predict the effect of changes in activity of enzymes, or changes in enzyme kinetic parameters, on sensitivity to inhibition. Kinetic simulation has provided insight into several aspects of the biochemical pharmacology of antimetabolites, including drug sensitivity and resistance, and drug-drug interactions. Two specific studies are described in detail. The first concerns the importance of the ratio of competing enzymes in determining the selectivity of inhibitors of one of the competing enzymes, studied by a simple model. The second case study examines the effect of alternative biosynthetic pathways, the de novo and salvage pathways of pyrimidine nucleotide biosynthesis, on the selectivity of antipyrimidine drugs, as studied by a detailed model of 27 reactions of pyrimidine metabolism.

Identification and quantitation of 1,4-butanediol in mammalian tissues: an alternative biosynthetic pathway for gamma-hydroxybutyric acid

Identification and quantitation of 1,4-butanediol in mammalian tissues: an alternative biosynthetic pathway for gamma-hydroxybutyric acid

Structural studies of the sugar chains of hen ovomucoid. Evidence indicating that they are formed mainly by the alternate biosynthetic pathway of asparagine-linked sugar chains.

The carbohydrate moieties of hen ovomucoid were released as oligosaccharides by hydrazinolysis. The neutral oligosaccharide fraction which comprised about 85% of the total sugar was fractionated into eight oligosaccharide fractions by Bio-Gel P-4 column chromatography. Occurrence of novel penta-antennary oligosaccharides in the larger three fractions was reported in the preceding paper (Yamashita, K., Kamerling, J.P., and Kobata, A. (1982) J. Biol. Chem. 257, 12809-12814). Structural studies of the remaining smaller oligosaccharides indicated that they all have Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc as their common core. The alpha-mannosyl residues occur either free or as one of the following five groups: GlcNAc beta 1 leads to 2Man, GlcNAc beta 1 leads to 4Man, GlcNAc beta 1 leads to 4(GlcNAc beta 1 leads to 2)Man, GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 2)Man, and GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 4)(GlcNAc beta 1 leads to 2) Man. In most oligosaccharides, a beta-N-acetylglucosamine residue is linked at the C-4 position of the beta-mannosyl residue of the core. The structural characteristic of the sugar chains of hen ovomucoid indicated that they are not formed by the ordinary processing pathway of the asparagine-linked sugar chains.