Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A. (1980) J. Biol. Chem. 255, 10777-10786). Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols. Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity. Consecutive modification experiments clearly show that the same active thiol is modified in both cases. However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence. Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence. The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence. Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence. This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.     

Metabolic effects of paraquat onSaccharomyces cerevisiae

Paraquat-tolerant cells ofSaccharomyces cerevisiae were selected by growing the cells in medium supplemented with paraquat either in a continuous selection culture with UV irradiation of the cells or in shaker flasks. When tranferred to fresh medium containing 1.0 mM paraquat, the selected cells showed growth rates and product formation patterns very similar to those of normal cells grown in a medium without paraquat. In the presence of paraquat, cells that had developed a tolerance were able to metabolize the fermentation products formed from glucose. Normal cells were unable to do so for considerable time.     

The effect of human bile on Bacteroides fragilis in health and disease

Bacteroides fragilis is generally believed to be stimulated by bile. Although B. fragilis is rarely found in the human duodenum, it is relatively frequent in gall bladder infections. To investigate this paradox, the growth of B. fragilis in the human bile in both health and disease was studied, and compared with the effect of bovine and porcine bile. B. fragilis was stimulated by the bovine bile and inhibited by the porcine bile. The normal human bile was either bacteriostatic or inhibitory whereas bile from cholelithiatic patients, in 50% of cases, stimulated the growth of B. fragilis. This explains the relative prevalence of B. fragilis in cholelithiatic cholecystitis patients although it is not a resident flora of the duodenum.     

The effect of thiols on the dehalogenation of 5-iodo and 5-bromouracil

The 5-iodo- and 5-bromo- analogs of uracil are dehalogenated in the presence of both cysteine and 2-mercaptoethanol to yield uracil. Presumably, the reaction involves the initial addition of the thiol group across the 5,6 double bond of the halopyrimidine to yield the corresponding 5-halo, 5,6-dihydrouracil-6-thioether which then dehalogenates to yield uracil. Under comparable conditions, cysteine causes more rapid dehalogenation of both halouracils than does 2-mercaptoethanol.Thiol containing compounds catalyze hydrogen-deuterium exchange at carbon five of uracil (1–3) and have been implicated as having a catalytic effect in the deamination of cytosine (4,5). Presumably, these reactions involve the reversible nucleophilic addition of the thiol group across the 5,6 double bond of the pyrimidine to yield the corresponding 5,6 dihydropyrimidine with a substituted thioether group on carbon six. This pathway is supported by comparable reactions involving the addition of bisulfite to the pyrimidine ring system (6–10). Different from the bisulfite addition compounds, the thioether containing dihydropyrimidine adducts have not been isolated and characterized; however, 5′-deoxy-5′,6-epithio-5,6-dihydro-2′,3′-0-isopropylideneuridine resulting from the intramolecular attack of the 5′ thiol group on carbon six of the uracil ring system of 5′-deoxy-5′-thio-2′,3′-0-isopropylideneuridine has been isolated and characterized (11).In a recent communication, we reported that bisulfite buffer systems catalyze the dehalogenation of 5-iodo-, 5-bromo-, and 5-chlorouracil (12). The object of this work is to demonstrate that cysteine and 2-mercaptoethanol, sulfur nucleophiles with more physiological importance than bisulfite, also cause halopyrimidine dehalogenation under nearly physiological conditions of temperature and pH.     

脆弱拟杆菌的研究进展

脆弱拟杆菌是定殖于哺乳动物肠道中的共生菌,同时也是临床感染病例中常见的条件致病菌。本文从致病及益生特性两大方面综述脆弱拟杆菌的研究现状,着重讨论了脆弱拟杆菌作为潜在益生菌在预防和治疗糖尿病及免疫性疾病中所起的重要作用,从而为筛选及应用益生脆弱拟杆菌菌株提供一定的参考。     

Nutritional Features of Bacteroides fragilis subsp. fragilis

Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B₁₂, minerals, bicarbonate-carbon dioxide buffer, NH₄Cl, and sulfide. The vitamin B₁₂ requirement of 0.1 ng/ml was replaced with 7.5 μg of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, β-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B₁₂, or related compounds, must be available during much of the time.     

The cell wall-associated inulinase of Kluyveromyces fragilis

The yeast Kluyveromyces fragilis (ATCC 12424) was grown on a 2% inulin-1% yeast extract medium for 36 h and subsequently fixed with 0.5% glutaraldehyde. The glutaraldehyde treatment did not affect the -fructofuranosidase (inulinase, EC 3.2.1.7) activity of the cells but it did make the cells resistant to chemical and physical treatments that normally release -fructofuranosidase from untreated cells. The enzyme in the treated cells exhibited K_m values for sucrose and raffinose identical to those obtained for the free enzyme. The cell wall of the treated cells exhibited the same diffusion properties for sucrose, raffinose, and inulin as those observed for untreated cells. The -fructofuranosidase was not bound covalently to the cell by the glutaraldehyde treatment. The results support the permeability barrier model for the enzyme retention in the yeast cell wall.     

The effect of coenzyme A and structurally related thiols on the mammalian fatty acid synthetase

A comparison was made of the structural features of thiol compounds which can interact with the mammalian fatty acid synthetase. Three functional characteristics were examined: (i) the ability of the free thiols, at low concentrations, to satisfy the essential thiol requirement of the enzyme, (ii) the ability of the free thiols, at higher concentrations, to inhibit enzyme activity, and (iii) the ability of the malonyl esters of these thiol compounds to act as substrates for fatty acid synthesis. The relative effectiveness of the various thiols studied was identical in all three roles. Coenzyme A and N-hexanoylcysteamine were the most effective, pantetheine and N-butyrylcysteamine were less effective, and N-acetylcysteamine was totally ineffective. These results lend strong support to our hypothesis (A. Stern, B. Sedgwick, and S. Smith, 1982, J. Biol. Chem.257, 799–803) that the various effects of CoA and structurally related thiols are localized at one and the same site, namely, the site of transfer of substrates between coenzyme A ester form and enzyme-bound form.     

The phosphonopyruvate decarboxylase from Bacteroides fragilis

The Bacteroides fragilis capsular polysaccharide complex is the major virulence factor for abscess formation in human hosts. Polysaccharide B of this complex contains a 2-aminoethylphosphonate functional group. This functional group is synthesized in three steps, one of which is catalyzed by phosphonopyruvate decarboxylase. In this paper, we report the cloning and overexpression of the B. fragilis phosphonopyruvate decarboxylase gene (aepY), purification of the phosphonopyruvate decarboxylase recombinant protein, and the extensive characterization of the reaction that it catalyzes. The homotrimeric (41,184-Da subunit) phosphonopyruvate decarboxylase catalyzes (kcat = 10.2 +/- 0.3 s-1) the decarboxylation of phosphonopyruvate (Km = 3.2 +/- 0.2 microm) to phosphonoacetaldehyde (Ki = 15 +/- 2 microm) and carbon dioxide at an optimal pH range of 7.0-7.5. Thiamine pyrophosphate (Km = 13 +/- 2 microm) and certain divalent metal ions (Mg(II) Km = 82 +/- 8 microm; Mn(II) Km = 13 +/- 1 microm; Ca(II) Km = 78 +/- 6 microm) serve as cofactors. Phosphonopyruvate decarboxylase is a member of the alpha-ketodecarboxylase family that includes sulfopyruvate decarboxylase, acetohydroxy acid synthase/acetolactate synthase, benzoylformate decarboxylase, glyoxylate carboligase, indole pyruvate decarboxylase, pyruvate decarboxylase, the acetyl phosphate-producing pyruvate oxidase, and the acetate-producing pyruvate oxidase. The Mg(II) binding residue Asp-260, which is located within the thiamine pyrophosphate binding motif of the alpha-ketodecarboxylase family, was shown by site-directed mutagenesis to play an important role in catalysis. Pyruvate (kcat = 0.05 s-1, Km = 25 mm) and sulfopyruvate (kcat approximately 0.05 s-1; Ki = 200 +/- 20 microm) are slow substrates for the phosphonopyruvate decarboxylase, indicating that this enzyme is promiscuous.     

The oxidation of thiols by ionizing radiations

Thiol compounds, such as glutathione, 2,3-dimercaptopropanol (BAL), propane-1,3-dithiol, and N-phenylaminopropanedithiol, were readily oxidized by x-rays, beta rays, and gamma rays. The ionic yield for this oxidation was about the same, 3 at pH 7, on irradiation with x-rays and with beta rays; it was 23 per cent less on irradiation with gamma rays. The ionic yield varied with the hydrogen ion concentration, increasing as the pH value increased. There was no reduction of oxidized glutathione on irradiation with dosages of x-rays and gamma rays which produced oxidation of the reduced compound. In the absence of oxygen, the oxidation of thiols by ionizing radiations was only 33 per cent of that obtained in the presence of dissolved oxygen. When the thiol solutions were irradiated in the presence of dissolved oxygen, catalase protected them from oxidation by 17 to 27 per cent.     

The influence of thiols on the pre-irradiation incubation effect of nitroimidazoles in E. coli cells

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0.15 mmol dm-3) completely protected against the pre-irradiation incubation effect of misonidazole (2.5 mmol dm-3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.     

The role of endogenous thiols in intrinsic radioprotection

Observations are reviewed from experiments performed to study the role of endogenous thiols in the radiation response of cells using a glutathione-deficient and a related glutathione-proficient cell strain. The effect of glutathione in the initial radical reactions was considered and the yield of single-strand DNA breaks was the end-point of the response. The rejoining of breaks and clonogenic survival were chosen as end-points when, in addition, the role of glutathione in the subsequent biochemical processes was studied. The results were interpreted to indicate that glutathione plays a role in both the radical and the biochemical reactions which follow irradiation. In the former case, it functions as a damage-restituting reactant, in general agreement with the 'competition model'. Some biochemical repair processes, in particular those concerned with the rejoining of breaks induced by radiation in the presence of oxygen or misonidazole, appear also to be critically dependent on glutathione. Due, probably, to its particular spatial distribution, endogenous glutathione is specific in the radical processes, and exogenous thiols cannot be substituted for it. No such specificity was indicated in the biochemical processes related to strand break rejoining.