Factors affecting counteraction by methylamines of urea effects on aldose reductase

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing Vmax or raising Km, yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the Km and Vmax of aldose reductase (EC 1.1.1.21), an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, betaine, and trimethylamine N-oxide), substrates (dl-glyceraldehyde and d-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that Vmax of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on Km are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of Km of wild-type enzyme by urea and/or methylamines that is partially additive, whereas at the other extreme we find that urea raises Km for d-xylose of the C298A mutant, betaine lowers the Km, and the two counteract in a classical fashion so that at a 2:1 molar ratio of betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme.

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66 → Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80–90°C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded β/α-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Inducible expression of exogenous genes in Dictyostelium discoideum using the ribonucleotide reductase promoter

We report here the development of a regulated gene expression system for Dictyostelium discoideum based on the DNA-damage inducibility of the rnrB gene. rnrB, which codes for the small subunit of the enzyme ribonucleotide reductase, responds to DNA-damaging agents at all stages of the D.discoideum life cycle. Doses that have little effect on development have previously been shown to increase the level of the rnrB transcript by up to 15-fold. Here we show that all elements necessary for DNA-damage induction are contained in a 450 bp promoter fragment. We used a fusion of the rnrB promoter with the gene encoding GFP to demonstrate an up to 10-fold induction at the RNA level, which appears in all aspects similar to induction of the endogenous rnrB transcript. Using a fusion with the lacZ gene we observed an up to 7-fold induction at the protein level. These results indicate that the rnrB promoter can be used to regulate the expression of specific genes in D.discoideum. This controllable gene expression system provides the following new characteristics: the induction is rapid, taking place in the order of minutes, and the promoter is responsive at all stages of the D.discoideum life cycle.

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd1 nitrite reductase from P. aeruginosa.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia

Low folate intake as well as alterations in folate metabolism as a result of polymorphisms in the enzyme methylenetetrahydrofolate reductase (MTHFR) have been associated with an increased incidence of neural tube defects, vascular disease, and some cancers. Polymorphic variants of MTHFR lead to enhanced thymidine pools and better quality DNA synthesis that could afford some protection from the development of leukemias, particularly those with translocations. We now report associations of MTHFR polymorphisms in three subgroups of pediatric leukemias: infant lymphoblastic or myeloblastic leukemias with MLL rearrangements and childhood lymphoblastic leukemias with either TEL-AML1 fusions or hyperdiploid karyotypes. Pediatric leukemia patients (n = 253 total) and healthy newborn controls (n = 200) were genotyped for MTHFR polymorphisms at nucleotides 677 (C→T) and 1,298 (A→C). A significant association for carriers of C677T was demonstrated for leukemias with MLL translocations (MLL+, n = 37) when compared with controls [adjusted odd ratios (OR) = 0.36 with a 95% confidence interval (CI) of 0.15–0.85; P = 0.017]. This protective effect was not evident for A1298C alleles (OR = 1.14). In contrast, associations for A1298C homozygotes (CC; OR = 0.26 with a 95% CI of 0.07–0.81) and C677T homozygotes (TT; OR = 0.49 with a 95% CI of 0.20–1.17) were observed for hyperdiploid leukemias (n = 138). No significant associations were evident for either polymorphism with TEL-AML1+ leukemias (n = 78). These differences in allelic associations may point to discrete attributes of the two alleles in their ability to alter folate and one-carbon metabolite pools and impact after DNA synthesis and methylation pathways, but should be viewed cautiously pending larger follow-up studies. The data provide evidence that molecularly defined subgroups of pediatric leukemias have different etiologies and also suggest a role of folate in the development of childhood leukemia.

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata1

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation1

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

12-Oxophytodienoate-10,11-Reductase: Occurrence of Two Isoenzymes of Different Specificity against Stereoisomers of 12-Oxophytodienoic Acid1

The reduction of 12-oxophytodienoic acid (OPDA) to 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of the isomer preference of OPR has indicated that the activity is composed of two isoenzymes exhibiting different stereoselectivities. The two isoforms of OPR have been separated, using protein extracts of Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported earlier from the same species and corresponding to the cloned OPR from Arabidopsis, utilized 9R,13R-OPDA >> 9S,13R-OPDA but not the 13S-configured isomers, whereas the new activity, OPRII, effectively reduced all four OPDA isomers, including the natural 9S,13S-OPDA (cis-[+]-OPDA). OPRII activity is characterized in detail. The enzyme's enzymatic, biochemical, and immunological properties prove that it is a close relative of OPRI. The roles of OPRI and OPRII in octadecanoid biology are discussed.

Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Regulation of Leaf Senescence by Cytokinin, Sugars, and Light : Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Characterization of the osmotic response element of the human aldose reductase gene promoter.

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.