D-threo-tetrahydrobiopterin is synthesized via 1'-oxo-2'-D-hydroxypropyl-tetrahydropterin in Dictyostelium discoideum Ax2

The biosynthesis of D-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of L-erythro-tetrahydrobiopterin (BH4) and 0.6% of DH4 productions in the wild type cells. The mutant cells were also identified to excrete D- and L-sepiapterin, which were presumed to originate from intracellular 1'-oxo-2'-D-hydroxypropyl- and 1'-oxo-2'-L-hydroxypropyl-tetrahydropterin (H4-pterin), respectively. Furthermore, in a coupled assay with Dictyostelium SR, the mutant cell extract exhibited a novel enzyme activity converting 6-pyruvoyltetrahydropterin to 1'-oxo-2'-D-hydroxypropyl-H4-pterin. These results are clear demonstration of the in vivo synthesis of DH4 via 1'-oxo-2'-D-hydroxypropyl-H4-pterin as well as an alternative synthesis of BH4 and DH4 in the complete absence of SR.     

Structure of rat aldose reductase-like protein AKR1B14 holoenzyme: Probing the role of His269 in coenzyme binding by site-directed mutagenesis

Rat aldose reductase-like protein (AKR1B14) is the ortholog of mouse vas deferens protein (AKR1B7) playing roles in detoxification of reactive aldehydes and synthesis of prostaglandin F_2α. The crystal structure of the binary complex (AKR1B14-NADPH) was determined at 1.86 Å resolution, and showed that the adenine ring and the 2′-phosphate group of the coenzyme formed π-stacking and electrostatic interactions with the imidazole ring and ND1 atom, respectively, of His269, which is not conserved in other aldose reductase-like proteins. The interactions were supported by site-directed mutagenesis of His269 to Arg, Phe and Met, which increased the K_m for NADPH by 4, 7 and 127-fold, respectively. This is the first report of the tertiary structure of a rodent AKR1B7 ortholog, which describes the role of a novel dual interaction for the non-conserved His269 in coenzyme binding.     

Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of L-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin

The ORF sequences of the gene encoding sepiapterin reductase were cloned from the genomic DNAs of Chlorobium tepidum and Chlorobium limicola, which are known to produce L-threo- and L-erythro-tetrahydrobiopterin (BH4)-N-acetylglucosamine, respectively. The deduced amino acid sequence of C. limicola consists of 241 residues, while C. tepidum SR has three residues more at the C-terminal. The overall protein sequence identity was 87.7%. Both recombinant proteins generated from Escherichia coli were identified to catalyze reduction of diketo compound 6-pyruvoyltetrahydropterin to L-threo-BH4. This result suggests that C. limicola needs an additional enzyme for L-erythro-BH4 synthesis to yield its glycoside. The catalytic activity of Chlorobium SRs also supports the previously proposed mechanism of two consecutive reductions of C1' carbonyl group of 6-pyruvoyltetrahydropterin via isomerization reaction.     

Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E. coli

A cDNA clone (SSC801) putatively encoding sepiapterin reductase (SR) was obtained from the expressed sequence tag clones of Dictyostelium discoideum. The cDNA sequence of 878 nucleotides constituted an ORF of 265 amino acid residues but was missing a few N-terminal residues. The deduced amino acid sequence showed 29.8% identity with mouse SR sequence and a molecular mass of 29,969 Da. The coding sequence was cloned in E. coli expression vector and overexpressed. The purified His-tag recombinant enzyme was confirmed to have the genuine activity of SR to produce tetrahydrobiopterin from 6-pyruvoyltetrahydropterin in a coupled assay with 6-pyruvoyltetrahydropterin synthase as well as dihydrobiopterin from sepiapterin. However, dictyopterin was not observed in our assay condition. The enzyme was also inhibited by N-acetylserotonin and to a lesser extent by melatonin. Km values for NADPH and sepiapterin were 51.8+/-2.7 microM and 40+/-2 microM, respectively. Vmax was determined as 0.14 micromol/min/mg of protein.     

Conversion of a NADPH-dependent aldehyde reducing enzyme into aldose reductase

• 1.1. Aldose reductase, aldehyde reductase and high-K_m, aldose reductase were purified from the inner medulla of dog kidney.
• 2.2. Compared with aldose reductase, high-K_m aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties.
• 3.3. High-K_m aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution.
• 4.4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC₅₀ values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-K_m aldose reductase.

     

Expression,Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.     

Mutations in the Sepiapterin Reductase Gene Cause a Novel Tetrahydrobiopterin-Dependent Monoamine-Neurotransmitter Deficiency without Hyperphenylalaninemia

Classic tetrahydrobiopterin (BH(4)) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH(4). Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC-->CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397-1401delAGAAC) resulting in abolished SPR-gene expression and an A-->G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH(4) biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH(4) in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH(4) and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.     

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.     

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling: RELATIVE IMPORTANCE OF THE DE NOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS*

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.In vascular disease states such as atherosclerosis and diabetes, endothelial nitric oxide (NO) bioactivity is reduced, and oxidative stress is increased, resulting in endothelial dysfunction. It has become apparent that enzymatic “coupling” of endothelial NO synthase by its cofactor tetrahydrobiopterin (BH4)² plays a key role in maintaining endothelial function. Indeed, the balance between NO and superoxide production by eNOS appears to be determined by the availability of BH4 versus the abundance of 7,8-dihydrobiopterin (BH2, that is inactive for NOS cofactor function and may compete with BH4 for NOS binding (1). Intracellular biopterin levels are regulated principally by the activity of the de novo biosynthetic pathway (Fig. 1). Guanosine triphosphate cyclohydrolase I (GTPCH; EC 3.5.4.16) catalyzes the formation of dihydroneopterin triphosphate from GTP, and BH4 is generated by two further steps through 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. GTPCH appears to be the rate-limiting enzyme in BH4 biosynthesis, and overexpression of GTPCH is sufficient to augment BH4 levels in cultured endothelial cells (2). Electron paramagnetic resonance spectroscopy studies have shown that BH4 both stabilizes and donates electrons to the ferrous-dioxygen complex in the oxygenase domain, as the initiating step of l-arginine oxidation (3–5). In this reaction BH4 forms the protonated trihydrobiopterin cation radical, which is subsequently reduced by electron transfer from NOS flavins. When BH4 availability is limiting, electron transfer from NOS flavins becomes uncoupled from l-arginine oxidation, eNOS generates superoxide rather than NO, BH4 becomes oxidized to catalytically incompetent BH2, and a futile feed-forward cascade of BH4 destruction proceeds (1). Recent studies reveal that BH4 and BH2 bind eNOS with equal affinity and that BH2 can efficiently replace eNOS-bound BH4, resulting in eNOS uncoupling (6). Indeed, we have previously shown that the relative abundance of eNOS versus BH4 together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling (7), a hypothesis supported by a recent publication where BH2 levels are elevated after exposure of bovine aortic endothelial cells to DHFR-specific siRNA (8). Thus, mechanisms that regulate the BH4:BH2 ratio independently of overall biopterin levels may play an equally important role in regulating eNOS coupling as the well established role of GTCPH, which regulates de novo BH4 biosynthesis. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR; EC 1.5.1.3) can reduce BH2, thus regenerating BH4 (9, 10). It is, therefore, likely that net BH4 bioavailability within the endothelium reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. In human liver extracts DHFR has been shown to reduce BH2 back to BH4 as part of the salvage pathway for biopterin synthesis (11). However, the role of this pathway and the extent to which it regulates intracellular BH4 levels in vivo remains unknown. Recent work by Chalupsky and Cai (2) investigated the functionality of endothelial DHFR in cultured bovine aortic endothelial cells. Exposure to angiotensin II down-regulated DHFR expression, decreased BH4 levels, and increased eNOS uncoupling, which was restored by overexpression of DHFR (2). A recent study also suggests that perturbation of BH4 metabolism differentially affects eNOS phosphorylation sites. Knockdown of DHFR by siRNA inhibits vascular endothelial growth factor-induced dephosphorylation of eNOS at Ser-116, an effect that is completely recovered by the addition of exogenous BH4 (8). However, the requirement for DHFR in regulating intracellular BH4 homeostasis and the quantitative relationships that relate BH4 de novo synthesis versus BH4 recycling to eNOS coupling remain uncertain. Accordingly, we sought to address these questions using both pharmacologic and genetic manipulation of DHFR activity and related these interventions to effects on eNOS coupling. We manipulated DHFR in both endothelial cells and in novel cell lines that stably express an eNOS-GFP fusion protein and where expression of human GTPCH can be regulated by doxycycline in order to test the effects of variations in intracellular BH4 biosynthesis (7). We report that although GTPCH is the key regulator of the total amount of intracellular biopterins, DHFR is critical to eNOS function by determining BH4:BH2 ratio and, thus, in maintaining eNOS coupling. In particular, DHFR is important in preventing “self-propagated” eNOS uncoupling in conditions of low total biopterin levels, when eNOS-dependent oxidation of BH4 that would further exacerbate eNOS uncoupling can be rescued by DHFR.Open in a separate windowFIGURE 1.Schematic representation of the BH4 recycling pathway and eNOS coupling. BH4 is synthesized from GTP via a series of reactions involving GTPCH, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase (SR) and DHFR. DHFR activity can be inhibited by MTX. GFRP, GTP cyclohydrolase feedback regulatory protein. PTPS, 6-pyruvoyl-tetrahydropterin synthase.     

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.     

Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr^−/− mouse model. Unexpectedly, the BH4 contents in the Qdpr^−/− mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr^−/− mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for a plasma membrane aldehyde reductase.

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPH-utilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoyl-CoA and stearoyl-CoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucose-induced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucose-induced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60-70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductase-like enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycol-Dextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltage-gated potassium channel beta subunits that contain aldehyde and aldose reductase-like NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237-243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

Biosynthesis and enantioselectivity in the production of the lilac compounds in Actinidia arguta flowers

Biosynthesis of the lilac alcohols and alcohol epoxides from linalool in ‘Hortgem Tahi’ kiwifruit (Actinidiaarguta) flowers was investigated by incubating flowers with rac-linalool, rac-[4,4,10,10,10-²H₅]linalool, (R)-8-hydroxylinalool and (R)-8-oxolinalool. All substrates were incorporated into the lilac alcohols although the (R)-configured compounds are not normally present in flowers. Biosynthesis of the lilac alcohol epoxides from rac-1,2-epoxy[4,4,10,10,10-²H₅]linalool and rac-[4′,4′, 8′, 8′,8′-²H₅]lilac aldehyde epoxide, rather than the lilac alcohols, was examined. Both substrates were non-enantioselectively converted to the lilac alcohol epoxides, suggesting two biosynthetic pathways for these compounds, contrary to previous reports. Their ability to process unnatural substrates indicates that A.arguta flowers have a greater biosynthetic capability than is suggested by their phytochemical composition. Linalool, the lilac compounds, and their biosynthetic intermediates were measured in the pistils, stamen, petals and sepals to determine if localisation in different organs contributed to only (S)-linalool being processed to the lilac compounds. Both linalool enantiomers were present in all organs, while most (97%) of the lilac compounds, and their precursors, were found in the petals. (S)-Linalool was not depleted from the flower petals, with respect to (R)-linalool, during the time of maximum production of the metabolites of (S)-linalool.     

HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators

Phytochelatins (PCs) are nonprotein peptides with the general structure (γ-Glu-Cys)_n-Gly (PC_n), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (γ-Glu-Cys-Gly) and/or previously synthesized PC_n as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5′-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.     

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

cAMP inhibits cytokine-induced biosynthesis of tetrahydrobiopterin in human umbilical vein endothelial cells

We studied the effects of cAMP on cytokine (interferon-gamma plus tumor necrosis factor-alpha)-induced stimulation of tetrahydrobiopterin (BH4) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH4 by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH4 by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH4 synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH4 by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH4 biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH4 by HUVEC.     

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-¹⁴C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K_m values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-l-methionine as a substrate, respectively.     

Sequence Analysis of a Novel Insertion Site of Transposon IS10

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′, which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5′-TGCTTGGTT-3′ instead of the previously reported 5′-NGCTNAGCN-3′. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome.