Michel Penninckx

A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses.

Glutathione (L-gamma-Glutamyl-L-Cysteinylglycine) appears as the major nonprotein thiol compound in yeasts. Recent advances have shown that glutathione (GSH) seems to be involved in the response of yeasts to different nutritional and oxidative stresses. When the yeast Saccharomyces cerevisiae is starved for sulfur or nitrogen nutrients, GSH may be mobilized to ensure cellular maintenance. Glutathione S-transferases may be involved in the detoxification of electrophilic xenobiotics. Vacuolar transport of metal derivatives of GSH ensure resistance to metal stress. Growth of methylotrophic yeasts on methanol results in the formation of an excess formaldehyde that is detoxified by a GSH-dependent formaldehyde dehydrogenase. Growth of yeasts on glycerol results in the accumulation of methylglyoxal detoxified by the glyoxalase pathway. Glutathione per se can react with oxidative agents or is involved in the oxidative stress response through glutathione peroxidase.

Metabolism and functions of glutathione in micro-organisms.

Publisher Summary The chapter discusses the biologically relevant chemistry of glutathione (GSH) and its occurrence in microbial cells. The GSH-related biochemical reactions and the physiological roles of GSH are summarized. The biosynthesis of GSH is remarkable in two ways: it is mRNA independent, and the glutamic residue is joined in an unusual peptide linkage of the γ -carbon atom to the cysteine residue. Due to this structural peculiarity, GSH is protected against proteolytic cleavage. The GSH status of cells is defined by the total concentration of GSH and the nature and distribution of the possible forms of occurrence of the tripeptide in the cell. GSH and related compounds are widespread in the microbial world, especially amongst organisms with an aerobic lifestyle. This observation emphasizes the role of GSH in cellular protection against by-products generated by oxidative metabolism, but it does not limit its functions to this role. Glutathione acts as an enzyme cofactor, transport component, nucleophilic substrate, and sulphur reservoir; and participates in key cellular processes such as protein synthesis and degradation, regulation of enzyme activity, synthesis of DNA, and maintenance of the integrity of cell membranes and organelles. Having a functional diversity, GSH is interrelated with a number of metabolic pathways and its intracellular modulation could have an impact on the entire cell, making it extremely difficult to associate directly a given cellular end-point with one molecule or system. Both in Escherichia coli and Saccharomyces cerevisiae, GSH plays an important role in cellular protection during chemical stresses in spite of the fact that key enzymes of detoxification, such as GSH peroxidase and GSH S-transferase, remain at a low level.

An important role for glutathione and gamma-glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast Saccharomyces cerevisiae.

When the yeast Saccharomyces cerevisiae sigma 1278b was starved for nitrogen, the total glutathione (GSH) pool increased from 7 to 17 nmol (mg dry wt)-1 during the first 2 h and then declined. More than 90% of the total GSH shifted towards the central vacuole during this time. This transient stimulation was not observed in the presence of buthionine-(S,R)-sulphoximine (BSO), a specific transition-state-analogue inhibitor of gamma-glutamylcysteine synthase (gamma-GCS), nor in a mutant strain deficient in this enzyme- gamma-Glutamyltranspeptidase (gamma-GT), a vacuolar enzyme responsible for the initial step of GSH degradation, was derepressed during nitrogen starvation. This mechanism can apparently enable the starved yeast cell to use the constituent amino acids from GSH which accumulate in the vacuole to satisfy its growth requirements for nitrogen.

Towards elucidation of the lignin degradation pathway in actinomycetes.

Six biodegradative actinomycete strains were grown on a dimeric model lignin compound of the β-aryl ether type. Although only two strains, Thermomonospora mesophila and Streptomyces badius, utilized the compound as a carbon and energy source and produced substantial amounts of monomeric products, all of the strains could demethylate the substrate and oxidize Cα on the phenylpropane side-chain. Streptomyces sp. EC1 produced small amounts of aromatic acids and unidentified lignin-derived products when grown on straw. This organism also produced cell-bound demethylase requiring H2O2 and Mn2+, protocatechuate 3,4-dioxygenase and β-carboxymuconate decarboxylase activity in response to growth on low-molecular-mass aromatic compounds but not lignocellulose or its polysaccharide components. Extracellular peroxidase and catalase activity were detected in all of the strains. These data are used to propose a scheme by which actinomycete attack of the lignin component of plant biomass can be envisaged.

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation. However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.

Regulation of the production of hemicellulolytic and cellulolytic enzymes by a Streptomyces sp. growing on lignocellulose.

A Streptomyces sp. isolated from compost degraded the hemicellulose fraction of straw efficiently but apparently not native cellulose. Ball-milled straw induced endoglucanase, beta-glucosidase, beta-xylanase and beta-xylosidase. Carboxymethylcellulose, cellotetraose and cellotriose induced cellulolytic enzymes specifically whereas cellobiose acted as inducer for beta-glucosidase only. Cellotriose and cellotetraose induced beta-glucosidase, but only partially induced endoglucanase. Hemicellulose (in the form of xylan) and xylobiose induced only beta-xylanase and beta-xylosidase. Kraft lignin and syringic acid induced beta-xylanase and endoglucanase but not the other enzymes. 3,4-Dimethoxycinnamic acid slightly induced beta-xylanase whereas 3,5-dimethoxy-4-hydroxycinnamic acid specifically induced endoglucanase. Neither veratric acid nor vanillic and ferulic acids induced any of the cellulolytic or hemicellulolytic enzymes. Enzyme production was subject to a form of carbon catabolite repression. Endoglucanase and beta-xylanase were excreted into the culture medium. Four protein components, one acidic (pI 5.2) and three basic (pI 8.15, 8.45 and 8.65) exhibited beta-xylanase activity. Two acidic components (pI 3.55 and 3.75) displayed endoglucanase activity.

gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione.

In the yeast Saccharomyces cerevisiae, the enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of gamma-GT displayed a K(m) (app) of 18+/-2 mM and a V(max) (app) of 48.5+/-5 nmol of GSH/min per mg of protein. Vacuoles containing gamma-GT displayed practically the same K(m), but a higher V(max) (app) (150+/-12 nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking gamma-GT showed no increase in V(max) (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H(+)) ATPase-coupled system. The V(max) (app)-increasing effect of gamma-GT concerns only the YCF1 system. gamma-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma-GT was also measured. It is proposed that gamma-GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying gamma-GT physiology and GSH metabolism.

Factor analysis of the relationships between several physico-chemical and microbiological characteristics of some Belgian agricultural soils

Abstract Twenty Belgian agricultural soils, 16 of which had been organically cultivated, were examined for their biochemical and microbiological properties. In spite of the very different nature of the characteristics studied, close relationships were found between soil respiration and glucose mineralization rates, biomass-C as measured by the fumigation-incubation method, and several enzyme activities, namely FDA-hydrolase, alkaline phosphatase and dehydrogenase. Correlation coefficients between urease activity and other biological measurements were always found to be endowed with a negative sign. Moreover, factor analysis of the data showed that some physico-chemical characteristics such as soil organic C, total N, clay content and CEC were closely related to most of the biological measurements, while pH and sand content were not. Two procedures for the determination of dehydrogenase activity and two methods of calculation of biomass-C were also compared.

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants.

The white-rot fungus Cerrena unicolor C-139 produced 450 000 U l(-1) of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K(m) value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.

Interaction between arginase and L-ornithine carbamoyltransferase in Saccharomyces cerevisiae. The regulatory sites of arginase.

The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control or arginine and ornithine, involves a regulatory site for arginine on the arginase distinct from its catalytic site. This regulatory site is responsible for the reinforcement effect of arginine on the inhibition of ornithine carbamoyltransferase by arginase. The binding site of ornithine carbamoyltransferase on arginase is also shown by our analysis.