Protein-engineering of Hydroperoxidases

Catalases (EC 1.11.1.6, hydrogen peroxide - hydrogen peroxide oxidoreductases) protect aerobic organisms against the toxic effects of hydrogen peroxide which they cleave into water and molecular oxygen. Typical catalases, forming the largest of three subgroups, are found in almost all aerobically respiring organisms, both prokaryotes and eukaryotes. These enzymes are homotetramers, 200-340 kDa in size with four prosthetic haem groups. The native quaternary structure of typical catalases is strictly required for maintaining their catalytic function. The crystal structures of several catalases of bacterial, fungal, or mammalian origin have been resolved and reveal an extremely well conserved “catalase fold” (Figure 1). This fold is characterised by two remarkable structural features:
Substrates have to enter the active site via the funnel-shaped main substrate channel, with a large cross-section at the molecule´s surface, and a narrow entrance to the distal haem side. This long, narrow and hydrophobic channel may prevent ready access to the active site for bulky or highly polar substrates. On the other hand, it is difficult to understand how the very high catalytic turnover of these enzymes (kcat=4x107M-1s-1, for human erythrocyte catalase) can be obtained under these conditions.
Secondly, there are extensive interactions between each pair of subunits, the most remarkable one being a unique pseudoknot formed by the extended N-terminal domain (N-arm) of one monomer which penetrates different sections of the so-called wrapping loop of two other subunits (Figure 2). Most likely this knot cannot be formed in the course of association of already folded monomers. In vivo dimerisation and eventually tetramerisation of only partially folded polypeptide chains will occur, followed by an ordered docking of the exchanged arms, and finally the wrapping loops holding them in place.

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Li, Sh., Peck-Radosavljevic, M., Koller, E., Koller, F., Kaserer, K., Kreil, A., Kapiotis, S., Hamwi, A., Weich, H.A., Valent, P., Dudczka, R., and Virgolini, I. (2001). Characterization of 123I-vascular endothelial growth factor-binding-sites expressed on human tumour cells: Possible implication for tumour scintigraphy. Int. J. Cancer 91, 789-796.

Li, Sh., Peck-Radosavljevic, M., Koller, E., Koller, F., Kaserer, K., Kreil, A., Kapiotis, S., Hamwi, A., Weich, H.A., Valent, P., Dudczka, R., and Virgolini, I. (2001). Characterization of 123I-vascular endothelial growth factor-binding-sites expressed on human tumour cells: Possible implication for tumour scintigraphy. Int. J. Cancer 91, 789-796.

Zamocky, M., Godocikova, J., Koller, F., and Polek, B. (2001). Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds. Anton Leeuw. Int. J. G. 79, 109-117.

Volf, I., Bielek, E., Moeslinger, Th., Koller, F., and Koller, E. (2000). Modification of the protein moiety of low density lipoprotein by hypochlorite generates a strong platelet agonist. Arterioscler. Thromb. Vasc. Biol. 20, 2011-2018.

Zamocky, M., Janecek, S., and Koller, F. (2000). Common phylogeny of catalase-peroxidases and ascorbate peroxidases. Gene 256, 169-182.