TOWARDS DESIGNER PEROXIDASES

Anette Henriksen1, Kåre Teilum2, Lars Østergaard2, Karen G. Welinder2 and Michael Gajhede1

1Protein Structure Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 København, Denmark.
2Department of Protein Chemistry, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 København, Denmark.

Keywords: Arabidopsis thaliana; Peroxidase structure; Anionic peroxidase; Neutral peroxidase;

Peroxidases have found widespread use in industry as an ingredient in immunochemical diagnostics, in decolorization and in dye transfer inhibition. To optimize peroxidases for these industrial uses and in general to aid rationalizing the structural determinants for peroxidase stability and catalytic activity, two peroxidases from Arabidopsis thaliana have been purified and crystallized.

The cationic horseradish peroxidase C, HRP C (1,2) has been a model compound for the study of pre-steady state kinetics and the characterization of high oxidation state intermediates in heme proteins (3). HRP C is 53% and 56% identical to neutral Arabidopsis thaliana peroxidase N, ATP N and anionic Arabidopsis thaliana peroxidase A2, ATP A2, respectively. A sequence identity of 94% is found between ATP A2 and horseradish peroxidase isoenzyme A2, HRP A2. The kinetic and redox-properties of HRP A2 is well characterized (4,5) and the enzyme is suggested to be more stable to auto-oxidation than HRP C (5).

ATP A2 and ATP N have been purified and refolded from Escherichia coli inclusion bodies in sub mg quantities. The purified protein was crystallized in hanging drops at 282 K using a sparse matrix test (6). The initial protein concentrations used were 1.0 mg/ml and 0.8 mg/ml for ATP A2 and ATP N respectively. Crystals used for X-ray experiments were selected from the matrix test on the basis of the possible cryo-protective properties of the mother liquor. For ATP N the mother liquor consisted of polyethyleneglycol 4000 and 2-propanol, while ATP A2 was crystallized from a solution containing Mg(CH3COO)2 and polyethyleneglycol 8000.

Synchrotron X-ray diffraction data at cryogenic temperature (100 K) were collected from single crystals at MAX-lab, Lund at beam line I711. Data were collected to 1.45 Å for ATP A2 and to 2.2 Å for ATP N. The structures were solved by molecular replacement with AmoRe (7). Horseradish peroxidase C (1) was the model structure for the molecular replacement. To our knowledge, the ATP A2 diffraction data collected has the highest resolution observed for any peroxidase. This rises possibilities for detailed studies of the heme environment, a region intensively studied by spectroscopic techniques.

  1. Gajhede, M., Schuller, D.J., Henriksen, A., Smith, A.T. and Poulos, T.L.: Crystal Structure of Horseradish Peroxidase C at 2.15 Å Resolution. Nat.Struct.Biol. 4 (1997) 1032-1038.

  2. Henriksen, A., Schuller, D.J., Meno, K., Welinder, K.G., Smith, A.T. and Gajhede, M.: Structural Interactions between Horseradish Peroxidase C and the Substrate Benzhydroxamic Acid Determined by X-ray Crystallography. Biochemistry, in press.

  3. Dunford, H.B. and Stillman, J.S.: On the Function and Mechanism of Action of Peroxidases. Coord. Chem. Rev. 19 (1976) 187-251.

  4. Kato, M., Aibara, S., Morita, Y., Nakatani, H. and Hiromi, K.: Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes. J Biochem (Tokyo) 95 (1984) 861-870.

  5. Yamazaki, I., and Nakajima, R.: Physico-Chemical Comparison Between Horseradish Peroxidases A and C. In Molecular and Physiological Aspects of Plant Peroxidases (Greppin, H., Penel, C. and Gaspar, Th., Eds.) University of Geneva Press (1986) pp. 71-84.

  6. Jancarik, J. and Kim, S.-H.: Sparse Matrix Sampling: A Screening method for Crystallization of Proteins. J. Appl. Cryst. 24 (1991) 409-411.

  7. Navaza, J.: AMoRe: an automated package for molecular replacement. Acta Crystallogr. A50 (1994) 157-163.