Biomedical subjects

Wilhelm Hansberg

Publications and source records attributed to Wilhelm Hansberg.

7 recordsLinked to original sources

PubMed2005

Functional and structural analysis of catalase oxidized by singlet oxygen.

Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.

Catalase↗

PubMed2005

Reactive oxygen species and development in microbial eukaryotes.

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. This raises questions about the pathways that different groups of organisms use to produce and sense ROS. In microbial eukaryotes, recent data show (i) increased ROS levels during cell differentiation, (ii) the existence of ROS-producing enzymes, such as NADPH oxidases (NOX), (iii) the involvement of NOX in developmental processes, and (iv) a conservation in the signal-transduction mechanisms used to detect ROS. This shows that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes and suggests that such strategy was selected early in evolution.

Antioxidants↗

PubMed2004

Unusual Cys-Tyr covalent bond in a large catalase.

Catalase-1, one of four catalase activities of Neurospora crassa, is associated with non-growing cells and accumulates in asexual spores. It is a large, tetrameric, highly efficient, and durable enzyme that is active even at molar concentrations of hydrogen peroxide. Catalase-1 is oxidized at the heme by singlet oxygen without significant effects on enzyme activity. Here we present the crystal structure of catalase-1 at 1.75A resolution. Compared to structures of other catalases of the large class, the main differences were found at the carboxy-terminal domain. The heme group is rotated 180 degrees around the alpha-gamma-meso carbon axis with respect to clade 3 small catalases. There is no co-ordination bond of the ferric ion at the heme distal side in catalase-1. The catalase-1 structure exhibited partial oxidation of heme b to heme d. Singlet oxygen, produced catalytically or by photosensitization, may hydroxylate C5 and C6 of pyrrole ring III with a subsequent formation of a gamma-spirolactone in C6. The modification site in catalases depends on the way dioxygen exits the protein: mainly through the central channel or the main channel in large and small catalases, respectively. The catalase-1 structure revealed an unusual covalent bond between a cysteine sulphur atom and the essential tyrosine residue of the proximal side of the active site. A peptide with the predicted theoretical mass of the two bound tryptic peptides was detected by mass spectrometry. A mechanism for the Cys-Tyr covalent bond formation is proposed. The tyrosine bound to the cysteine residue would be less prone to donate electrons to compound I to form compound II, explaining catalase-1 resistance to substrate inhibition and inactivation. An apparent constriction of the main channel at Ser198 lead us to propose a gate that opens the narrow part of the channel when there is sufficient hydrogen peroxide in the small cavity before the gate. This mechanism would explain the increase in catalytic velocity as the hydrogen peroxide concentration rises.

Amino Acid Sequence↗

PubMed2003

Cu,Zn-superoxide dismutase of Saccharomyces cerevisiae is required for resistance to hyperosmosis.

Here we analyzed the role of the antioxidant response in Saccharomyces cerevisiae adaptation to hyperosmotic stress. We show that Cu,Zn-superoxide dismutase (SOD1) plays a fundamental role in this adaptation process since under hyperosmosis SOD1 mutants lead to high protein oxidation levels and show a sensitive phenotype, which is reversed by the addition of N-acetylcysteine to the medium. Pretreatment with MnCl(2), a superoxide scavenger, improves the survival of the sod1 strain upon hyperosmosis. Additionally, we show that upon hyperosmotic shock there is a small and transient increase in SOD1 transcript levels, regulated by the protein kinase A-cAMP and SKN7 pathways.

Adaptation, Physiological↗

PubMed2003

Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

Animals↗

PubMed2002

Regulation and oxidation of two large monofunctional catalases.

The two Neurospora crassa catalase genes cat-1 and cat-3 were shown to encode Cat-1 and Cat-3 large monofunctional catalases. cat-1 and cat-3 genes are regulated differentially during the asexual life cycle and under stress conditions. A stepwise increase in catalase activity occurs during conidiation. Conidia have 60 times more catalase activity than exponentially growing hyphae. Cat-1 activity was predominant in conidia, during germination and early exponential growth. It was induced during prestationary growth and by ethanol or heat shock. Cat-3 activity was predominant during late exponential growth and at the start of the conidiation process. It was induced under stress conditions, such as H(2)O(2), paraquat, cadmium, heat shock, uric acid, and nitrate treatment. In general, Cat-1 activity was associated with nongrowing cells and Cat-3 activity with growing cells. The Cat-3 N-terminus sequence indicates that this catalase is processed and presumably secreted. Paraquat caused modification and degradation of Cat-1. Under heat shock both Cat-1 and Cat-3 were modified and degraded and Cat-1 was resynthesized. Paraquat and heat shock effects were observed only in the presence of air and are probably related to in vivo generation of singlet oxygen. Purified Cat-3 was modified with a photosensitizing reaction in which singlet oxygen is produced.

Aerobiosis↗

PubMedDate not supplied

Neurospora crassa catalases, singlet oxygen and cell differentiation.

The morphogenetic transitions of the N. crassa asexual life cycle are responses to a hyperoxidant state in which probably singlet oxygen is generated. Induction of catalase activity and catalase oxidation by singlet oxygen are consequences of this recurrent hyperoxidant state. Here the biochemical properties and regulation of two large monofunctional catalases are reviewed, and a new catalase-peroxidase gene and activity is described. Catalase-3 is associated to growing and Catalase-1 to non-growing cells. Under stressful conditions one of these catalases is synthesized, depending on whether growth can be continued or a resistant cell has to be made. The catalase-peroxidase Catalase-2 was possibly derived from a bacterial enzyme. In contrast to the other catalases, Catalase-2 had catalase and peroxidase activity. Catalase-2 was expressed under conditions in which vacuolization of hyphae is observed. All three enzymes have a chlorin in its active site instead of ferroprotoheme IX and are resistant to molar concentrations of hydrogen peroxide. These and all other catalases tested so far are oxidized by singlet oxygen, probably at the heme moiety. The catalase activity is virtually unaffected by oxidation, but the enzymes are probably degraded more rapidly than the unmodified ones.

Catalase↗