Sulfur metabolism enzymes in thermoacidophilus bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur:Fe(III) oxidoreductase, and sulfite:Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus strains N1 and SSO. Enzyme activity was revealed in cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.     

Structural insights into dioxygen-activating copper enzymes

Copper-containing enzymes that react with O(2) play a key role in many biological processes. Mononuclear, dinuclear and trinuclear copper centers function in O(2) binding, activation and subsequent substrate oxidation. Recent advances in the structural biology of O(2)-activating copper enzymes range from the identification of novel copper centers, such as that of particulate methane monooxygenase, to the elucidation of the details of O(2) binding and reactivity in peptidylglycine alpha-hydroxylating monooxygenase. Structures of phenoxazinone synthase and Fet3 contribute to our understanding of multicopper oxidases. Additionally, details of the tyrosinase structure provide new insight into how dicopper sites confer substrate specificity. A common theme for each of these enzymes is that the protein scaffold plays a major role in dictating the overall function.     

Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.     

Reduction of dioxygen by enzymes containing copper

The reduction of dioxygen is a key step in many important biological processes including respiration and ligand oxidation. Enzymes containing either iron or copper or, indeed, both elements are often involved in this process, yet the catalytic mechanisms employed are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. The aim of this article is to highlight the current structural knowledge regarding the process of dioxygen reduction using examples of copper-containing enzymes.     

铜胁迫对甘蔗生长及抗氧化酶活性的影响

甘蔗是重要的糖能兼用植物,同时也是潜在的重金属土壤修复植物。为了探明甘蔗对铜胁迫的生理响应,该研究采用营养液培养的方法分析了铜胁迫对甘蔗生长、叶片相对电导率、叶绿素含量及根系抗氧化酶活性等的影响。结果表明:甘蔗地上部分生物、叶绿素含量随铜浓度增加而下降,地上部分铜含量和叶片的相对电导率则随铜浓度增加而提高。根系的丙二醛(MDA)含量在胁迫24h开始发生显著的变化,在400μmol·L^-1铜处理下比对照显著增加了25.5%,随着胁迫时间的延长,丙二醛含量增加幅度上升。到胁迫72h,100μmol·L^-1铜处理的MDA含量也显著的提高。根系SOD、POD和CAT酶活性在胁迫24h开始发生显著的变化,但不同酶活性变化存在差异。SOD酶活性除了在胁迫的72h随铜浓度增加而提高,其他时间表现为在100μmol·L^-1铜处理下活性下降,随着铜浓度继续增加SOD酶活性提高,到400μmol·L^-1铜处理SOD酶活性又出现下降。POD酶活性在处理的24、48和72h表现为随浓度增加先提高后下降的趋势,但在处理7d时随铜浓度增加而提高。CAT酶活性随着浓度的升高和胁迫时间的延长而下降,到了胁迫的第7天,三个铜处理下CAT酶活性分别下降了89.98%、96.88%和98.50%。由此可见,铜胁迫严重影响甘蔗的生长,较短时间的处理就已经引起了甘蔗根系的氧化胁迫,根系中SOD、POD和CAT酶活性的变化在甘蔗对抗铜引起的氧化胁迫中起到重要作用。     

Canine models of copper toxicosis for understanding mammalian copper metabolism

Hereditary forms of copper toxicosis exist in man and dogs. In man, Wilson's disease is the best studied disorder of copper overload, resulting from mutations in the gene coding for the copper transporter ATP7B. Forms of copper toxicosis for which no causal gene is known yet are recognized as well, often in young children. Although advances have been made in unraveling the genetic background of disorders of copper metabolism in man, many questions regarding disease mechanisms and copper homeostasis remain unanswered. Genetic studies in the Bedlington terrier, a dog breed affected with copper toxicosis, identified COMMD1, a gene that was previously unknown to be involved in copper metabolism. Besides the Bedlington terrier, a number of other dog breeds suffer from hereditary copper toxicosis and show similar phenotypes to humans with copper storage disorders. Unlike the heterogeneity of most human populations, the genetic structure within a purebred dog population is homogeneous, which is advantageous for unraveling the molecular genetics of complex diseases. This article reviews the work that has been done on the Bedlington terrier, summarizes what was learned from studies into COMMD1 function, describes hereditary copper toxicosis phenotypes in other dog breeds, and discusses the opportunities for genome-wide association studies on copper toxicosis in the dog to contribute to the understanding of mammalian copper metabolism and copper metabolism disorders in man.     

Zinc,copper, and zinc- or copper-dependent enzymes in human hypertension

Imbalance of zinc and copper status has been hypothesized in human hypertension. A case-control study was carried out to elucidate the possible relationship between zinc and copper status and essential hypertension. Thirty-one subjects affected by mild stable hypertension, pharmacologically untreated, were investigated together with 31 normotensive controls individually matched for sex, age, and smoking habits. Zinc and copper in serum and urine were measured, and serum activities of alkaline phosphatase (AP), lactic dehydrogenase (LDH), copper-zinc superoxide dismutase (Cu?Zn SOD), lysyl oxidase (LOX), and monoamine oxidase (MAO) were evaluated. No significant difference in serum and urine zinc and copper content as far as in serum activity of zinc (AP and LDH) or copper (Cu?Zn SOD, LOX, and MAO)-dependent enzymes was found between hypertensives and normotensives. Positive relationships were found in normotensives between serum and urine levels of zinc (r=0.577;p=0.001) and copper (r=0.394;p=0.028), and between serum copper and Cu?Zn SOD (r=0.534;p=0.002). In normotensives, diastolic blood pressure and serum zinc were positively related (r=0.370;p=0.041). In hypertensives, inverse correlations were observed between diastolic blood pressure and AP (r=?0.498;p=0.004) and Cu?Zn SOD (r=?0.452;p=0.011), and between systolic blood pressure and LOX (r=?0.385;p=0.033). Diastolic blood pressure was related to LDH inversely in hypertensives (r=?0.357;p=0.049) and positively in normotensives (r=0.457;p=0.010). In normotensives, diastolic blood pressure was inversely related with MAO (r=?0.360;p=0.046). These findings support the hypothesis that an imbalance of zinc and copper status might be involved in human hypertension.     

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.     

Functional models for mononuclear nonheme iron enzymes

Increasing interest in mononuclear nonheme iron enzymes that activate dioxygen has resulted in an explosion of information on such enzymes in recent years. Concomitantly, efforts to model the active sites of these enzymes have produced synthetic complexes capable of mimicking some aspect of the reactivity of the metal center in several enzymes. These functional models carry out oxidative transformations analogous to those catalyzed by the enzymes and in some cases allow iron(III)-peroxo or iron(IV)-oxo intermediates to be trapped and characterized.     

Basic models for differential inhibition of enzymes

The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as “intra-site differential inhibition” and the molecules able to exert this action as “differential inhibitors”. This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate.     

Neurotoxic effects of copper: Inhibition of glycolysis and glycolytic enzymes

The effects of Cu²⁺ on glycolysis and several glycolytic enzymes were studied in rat brain extracts in vitro. At concentrations reportedly found in Wilson's disease, Cu²⁺ significantly inhibited lactate production from glucose or glucose-6-phosphate in rat brain postnuclear supernatant with an IC₅₀ of about 3 M. Cu²⁺ also inhibited several glycolytic enzymes. Amongst the latter, Cu²⁺ was most effective in inhibiting hexokinase (IC₅₀ for Cu²⁺=7 M), moderately effective in inhibiting pyruvate kinase (IC₅₀ for Cu²⁺=56 M), but least effective in inhibiting lactate dehydrogenase (IC₅₀ for Cu²⁺=300 M). These results suggest that inhibition of brain glycolysis may have pathophysiological importance in copper poisoning and in Wilson's disease.     

Conformational analysis and hydration models of copper threoninates and isoleucinates

The Consistent Force Field (CFF) method of conformational analysis was applied to ionic copper complexes with naturally occurring and allo-isomers of isoleucine and threonine, namely, [Cu(Thr)₂H_?2]^?2, (Cu(Ile)]⁺, and (Cu(Thr)]⁺ Hydration stabilization by means of a hydration shell model, as well as the effect of ligated water, were also computed. The difference in stability between the complexes of naturally occurring and allo-isoleucine is in accordance with experimental data. The theoretical results on [Cu(Thr)]⁺ complexes suggest a possible role of specific intramolecular interactions in determining the observed stability difference.     

Steady-state kinetics of models of respiratory chain enzymes with isopotential pools and conformational site enzymes.

The formal kinetics of the respiratory chain enzymes is a very complicated mathematical problem. Our main objective here is to introduce methodology that should be useful in approaching this problem, especially at steady state. Most of the work reported here is on models that involve one or two isopotential pools of enzymes and also possibly a site enzyme between two pools that may or may not undergo a conformation change. Preliminary topics treated are the “cross-over” phenomenon and the “mass action” approximation.     

Viscoelastic models for enzymes with multiple conformational states

In this article we represent an enzyme capable of exhibiting more than one conformational state as a viscoelastic unit embedded in a fluid medium. We show how this viscoelastic unit is thermally activated to make transitions between equilibrium states, and propose this model as a mesoscopic representation for transitions between conformational states of an enzyme. In this representation, kinetic constants for transitions between conformations are given explicitly in terms of interactions between the enzyme and the medium. Two applications of this model are discussed: mnemonic enzymes, and co-operative enzymes exhibiting half-of-the-sites reactivity.     

Thylakoid‐bound and stromal antioxidative enzymes in wheat treated with excess copper

Copper is a catalyst in the formation of reactive free radicals and its toxicity may be due, at least in part, to oxidative damage. The response of thylakoid‐bound and stromal antioxidative enzymes against the generation of superoxide radical was investigated in seedlings of wheat ( Triticum durum L. cv. Adamello) grown in hydroponic culture for 10 days and subjected to 10 and 50 µ M copper treatments. Electron spin resonance of roots evidenced a spectrum of copper, the intensity of which increased with the treatment, whereas the carbon‐centered free radical spectrum detected in the control leaves was not seen anymore in the treated samples. As well as thylakoids, photosystem II (PSII) particles were able to produce the superoxide radical. Increased superoxide production both by thylakoids and PSII was observed in the sample treated with 50 µ M Cu. Induction of thylakoid‐bound and stromal antioxidative enzymes, with the exception of dehydroascorbate reductase, was also detected in leaves treated with the highest copper concentration. No Mn‐superoxide dismutase (SOD, EC 1.15.1.1) was detected in thylakoids of wheat. Both stromal and thylakoid‐bound SOD were CuZn‐SOD with 16.2‐kDa subunits. Both western blotting and immuno‐electron microscopy showed that the SOD subunit was recognized by a polyclonal antibody against glyoxisomal CuZn‐SOD from watermelon cotyledon. In the stroma of wheat, ascorbate peroxidase showed at least three well‐resolved bands differently induced by copper treatments.