Mapping of quantitative trait loci (QTL) associated with activity of disulfide reductase and lipoxygenase in grain of bread wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

Activity of two enzymes of thiol-disulfide cell metabolism, lipoxygenase (LOX, EC 1.13.11.12) and disulfide-reductase (TPDO, EC 1.8.4.2) was studied in recombinant inbred lines of bread wheat ITMI. Their activity in the caryopsis may be connected with the gluten quality, one of the most important traits significant for breeding. The activity of lipoxygenase under favorable and droughty environmental conditions was shown to be associated with the quantitative trait locus (QTL) located on chromosome 4BS near the structural gene of a subunit of this enzyme. However, no QTL common to this enzyme and any characteristic of gluten quality have been found. Four loci responsible for the activity of disulfide reductase were identified on chromosomes 4A, 5D, 6A, and 7D. Previously, indicators of grain and flour properties, such as elasticity, flour strenght, and grain hardiness were mapped at the same loci. This indicates that the given enzyme participates in the formation of the protein complex upon maturation of wheat grain. The detected QTL can be involved in further genetic studies designed to establish the regularities of gluten formation.     

Identification,characterization, and comparison of RNA-degrading enzymes of wheat and barley

RNA-degrading enzymes play an important role in regulating gene expression, and sequence analyses have revealed significant homology among several plant RNA-degrading enzymes. In this study we surveyed crude extracts of the above-ground part of the common wheat (Triticum aestivum L.) and the cultivated barley (Hordeum vulgare L.) for major RNA-degrading enzymes using a substrate-based SDS-PAGE assay. Fifteen wheat and fourteen barley RNA-degrading enzymes, with apparent molecular masses ranging from 16.3 to 40.1 kD, were identified. These RNA-degrading enzymes were characterized by their response to pH changes and addition of EDTA and ZnCl₂ to the preincubation or incubation buffers. The 33.2- to 40.1-kD wheat and barley, 31.7-kD wheat, and 32.0-kD barley enzyme activities were inhibited by both zinc and EDTA and were relatively tolerant to alkaline environment. The 22.7- to 28.2-kD enzymes were inhibited by zinc but stimulated by EDTA. The 18.8-kD enzyme exists in both wheat and barley. It was active in an acid environment, was inhibited by zinc, but was not affected by EDTA. Two enzyme activities (31.0 and 32.0 kD) are unique to the common wheat. Contribution from Agriculture Research Division, University of Nebraska, Journal Series No. 9895.     

Improvement of the enzymatic activity of the hyperthermophilic cellulase from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).     

Mapping of quantitative trait loci (QTL) associated with activity of disulfide reductase and lipoxygenase in grains of durum wheat Triticum aestivum L. seeds

Activity of two enzymes of thiol-disulfide cell metabolism, lipoxygenase (LOX, EC 1.13.11.12) and disulfide-reductase (TPDO, EC 1.8.4.2) was studied in recombinant inbred lines of common wheat ITMI. Their activity in the caryopsis may be connected with the gluten quality, one of the most important traits significant for selection. The activity of lipoxygenase under favorable and droughty environmental conditions was shown to be associated with the quantitative trait locus (QTL) located on chromosome 4BS near the structural gene of a subunit of this enzyme. However, no QTL common to this enzyme and any characteristic of gluten quality have been found. Four loci responsible for the activity of disulfide reductase were identified on chromosomes 4A, 5D, 6A, and7D. Previously, indicators of grain and flour properties, such as elasticity, flour vigor, and grain hardiness were mapped at the same loci. This indicates that the given enzyme participates in the formation of the protein complex upon maturation of wheatgrain. The detected QTL can be involved in further genetic studies designed to establish the regularities of gluten formation.     

Location and prokaryotic expression of low molecular mass glutanin subunit gene <Emphasis Type="Italic">177-21</Emphasis> from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

To characterize the low molecular mass glutenin subunit gene 177-21 (AY994364) in wheat (Triticum aestivum L. cv. Jinan 177), we developed a specific PCR primer set to decide its locus with nullisomic-tetrasomic lines of Chinese spring wheat. The result showed that it was assigned to Glu-D3. The DNA fragment of 177-21 was then subcloned into the pGEX-4T-1 expression vector and expressed in E. coli with isopropyl-1-thio-β-D-galactoside induction. The result indicated that this gene encodes about 30 kD polypeptide and deduced amino acid sequence consists of eight cysteine residues. Of the eight, six may be related with the formation of intra-molecular disulfide bonds, the last two with the formation of inter-molecular disulfide bonds, which could be a potential extender in “glutenin polymer” to have positive influence on quality of wheat flour.     

Evidence for the proximity of two sulfhydryl groups at the active site of 6-phosphogluconate dehydrogenase

The reaction between 6-phosphogluconate dehydrogenase from Candida utilis and 5,5′-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH groups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.     

Introduction of a disulfide bridge enhances the thermostability of a <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> xylanase mutant

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.     

Sucrose synthase from wheat leaves. Comparison with the wheat germ enzyme

Sucrose synthase (UDP glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) was partially purified from wheat ( Triticum aestivum L. cv. San Agustin INTA) leaves and its properties compared with the wheat germ enzyme. The leaf enzyme moved faster in polyacrylamide gel electrophoresis, was more sensitive to SH reagents and crossreacted more slowly with antibody prepared towards the germ enzyme. Kinetic constants were of the same order for all substrates. UDP was a strong inhibitor of the synthesis reaction. MgCl₂ stimulated this reaction and partially reversed UDP inhibition. Molecular weight determined by gel filtration was 380 and 370 kdalton for the leaf and germ enzymes respectively. Both enzymes presented forms of higher molecular weight estimated to around 800 and 1000 kdalton. Neither sucrose synthase from leaves nor from germ were affected by fructose 6-P, fructose 1,6—P₂, glucose 1—P, glucose 6—P, fructose 2,6—P₂ and cAMP.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

Chloramphenicol effects on chlorophyll degradation and photosystem I assembly in the chlorina CD3 wheat mutant

We previously reported that applications of chloramphenicol to the chlorina wheat mutant, CD3, decreased the leaf Chl a/b ratio and enhanced accumulations of LHC proteins and LHC complexes during greening (Duysen et al. 1985). We have now examined Chl degradation and the change in Chl a/b ratios in wheat leaves kept in the dark as a measure of LHC destruction. Chl b was stable in chloroplasts of the CD3 wheat kept in darkness up to 5 days. Chloramphenicol significantly increased Chl b accumulations and impaired Chl a degradation in both CD3 mutant and normal wheat relative to untreated plants. Our Chl data suggest that the chloramphenicol induced accumulation of the LHC complex in the mutant wheat results from enhanced processing of LHC into the membrane rather than impairment of LHC degradation. The photosystem I (PSI) fraction of the CD3 wheat mutant was examined relative to that of normal wheat after 3 days greening. PSI was deficient in 25, 26, 26.5 kD LHCI protein in the mutant but both wheats accumulated low quantities of the 27–29 kD LHCII protein as detected by Western blot analysis. Chloramphenicol enhanced accumulations of several LHCI proteins primarily near 25 kD in the mutant and the 27–29 kD LHCII protein in normal wheat. The fluorescence emission and absorbance spectra suggest that chloramphenicol enhances accumulations of dissociated LHC in the PSI preparation of normal and CD3 mutant wheat.A contribution of North Dakota Agricultural Experiment Station. Published with approval of the Director as Journal Paper Number 1563.     

Effect of modification of sulfhydryl groups in soybean beta-amylase on the interaction with substrate and inhibitors

Methyl 2,4-dinitrophenyl disulfide (MDPS) is shown to be an effective methanethiolating reagent for sulfhydryl groups in proteins via thiol-disulfide exchange reaction. It reacts with the two reactive sulfhydryl groups (SH1 and SH2) in soybean beta-amylase. A decrease of the enzymatic activity accompanies the methanethiolation of SH2. After complete methanethiolation of SH2, the modified enzyme still has 9% of the initial activity. Modification of SH2 with cyanide and iodoacetamide reduces the enzymatic activity to 65 and 2% of the initial activity, respectively. Apparently, the residual activity depends upon the size of the substituent at SH2. The modified enzymes still have the almost same Km values for amylopectin and Kd values for enzyme-maltose and enzyme-cyclohexaamylose complexes as the native enzyme. In contrast to maltose and cyclohexaamylose, the Kd value of the enzyme-glucose complex increases in the order of cyanide-, MDPS-, and iodoacetamide-modified enzymes, indicating that SH2 is located near the binding site of glucose. It is proposed from the subsite structure of soybean beta-amylase that the position of SH2 and the glucose binding site is around subsite 1, where the nonreducing ends of the substrate bind productively.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S₂) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)₂] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)₂ serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)₂ is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S₂ by light and ferrodoxin; Trx-(SH)₂ is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control.
Thioredoxin-negative mutants ( trxA ) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains.

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.     

The Protein Disulfide Isomerase gene family in bread wheat (<Emphasis Type="Italic">T. aestivum L</Emphasis>.)

Background  

The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.     

Effect of traumatic acid on antioxidant activity in <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> (Chlorophyceae)

The present study was undertaken to test the influence of exogenously applied traumatic acid (TA) upon the activity of several antioxidant enzymes as well as lipid and protein peroxidation in green algae Chlorella vulgaris. Treatment with TA in concentration range of 10⁻⁶–10⁻⁵ M resulted in an increase of antioxidant enzyme (sodium dismutase, catalase, ascorbate peroxidase, NADH peroxidase, glutathione reductase) activity. Moreover, TA suppressed lipid peroxidation and oxidative destruction of proteins belonging to the SH groups. This data suggest that TA plays an important role in the metabolism of C. vulgaris and probably in its high ability to adapt to various environmental stress factors.     

Secreted alpha amidating enzymes are generated by specific posttranslational processing of precursors containing transmembrane domains

The biosynthesis and secretion of alpha amidating enzymes from CA-77 cells has been investigated to determine the relationship among the various forms of alpha amidating enzyme seen after purification of alpha amidating enzyme activity from conditioned cell culture media. Initially 2 proteins of 104 kD and 94 kD are synthesized. With time the 104 kD precursor is processed to 41 kD and 43 kD, and the 94 kD precursor is processed to 75 kD. The 41 kD, 43 kD, and 75 kD proteins are secreted into the medium as functional enzymes. In comparing these data with known cDNA sequence for alpha amidating enzyme we conclude that the 104 kD and 94 kD precursors are membrane bound proteins which are posttranslationally processed to generate secreted alpha amidating enzyme.     

Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free —SH groups. This allowed the oriented conjugation of alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.     

Extracellular Hemicellulolytic Enzymes from the Maize Endophyte <Emphasis Type="Italic">Acremonium zeae</Emphasis>

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and β-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars. Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica

PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.     

The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. Probes of conformational changes

4-Aminobutyrate aminotransferase is inactivated by preincubation with iodosobenzoate at pH 7. The reaction of 2 SH residues/dimer resulted in formation of an oligomeric species of Mr = 100,000 detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunits cross-linked via a disulfide bond are dissociated by addition of 2-mercaptoethanol which also restores full catalytic activity (Choi, S. Y., and Churchich, J.E. (1985) J. Biol. Chem. 260, 993-997). The substrate 2-oxoglutarate prevents inactivation of the enzyme by iodosobenzoate and the subsequent formation of one disulfide bond, whereas 4-aminobutyrate has no effect on the reactivity of SH groups with iodosobenzoate. Modified 4-aminobutyrate aminotransferase (containing 1 disulfide bond) catalyzes a half-transamination reaction; but it is unable to react with 2-oxoglutarate to generate the aldimine form of the enzyme. The spectroscopic properties (fluorescence yield and polarization of fluorescence) of PMP bound to the modified enzyme are different from those of pyridoxamine phosphate (PMP) bound to the native enzyme. The polarization of fluorescence values of PMP bound to the cross-linked enzyme, excited over the spectral range 310-370 nm, are greater (25%) than those of the cofactor of the native enzyme. An increase in the polarization values implies that the motion of PMP is restricted when the subunits are cross-linked via a disulfide bond.