The peroxisomal catalase A from S.cerevisisae is a representative typical tetrameric haem-containing catalase with known 3D structure [1]. It efficiently protects the cells from the deleterious effects of the catabolic by-product hydrogen peroxide. Our project of catalase-engineering focuses on the expression of variants with increased peroxidatic activity towards various aliphatic and aromatic substrates; additionally we attempt to uncover the intricate pathway of assembly which leads to the formation of pseudo-knot like structures by arm-exchange between neighboring subunits.

Single or double amino acid replacements were performed in the corresponding gene CTA1 with the quick change mutagenesis kit from Stratagene. After overexpression in yeast the engineered catalases were purified by two affinity chromatography steps. The catalatic and peroxidatic activities were determined according to standard protocols [2] and the eventual formation of inter-subunit disulfide bridges was monitored by SDS PAGE followed by western blot as described in [3].

We report a 12 to 1900-fold increase of the rate of 2-electron peroxidations of short chain alcohols for single exchange mutants V111S and V111T and an up to 70-fold increase for the 1-electron peroxidation of ABTS compared with wild type catalase A.

The double cysteine mutant L46C-Q349C shows 23% of the wild type catalatic activity, and under non-reducing conditions the formation of a dimer of 115 kDa was detectable. Currently we try to increase the catalytic efficiency of several double cysteine mutants by directed evolution (DNA shuffling).We also assay the unfolding stability of the respective mutants in the major substrate channel as well as those capable of disulfide bridge formation.

Acknowledgments: Part of this work was supported by grant #13637 of the Austrian Science Foundation (FWF).

[1] Mate, M.J., Zamocky, M., Nykyri, L.M., Herzog, C., Alzari, P.M., Betzel, C., Koller, F., Fita, I. , J. Mol. Biol., 286,135-149, 1999.

[2] Zamocky, M., Herzog, C., Nykyri, L.M., Koller, F., FEBS Lett., 367, 241-245, 1995.

[3] van den Burg, B., Dijkstra, B.W., Vinne, B., Stulp, B.K., Eijsink, G.H., Venema, G., Protein Engin., 6, 521-527, 1993.