Franz Koller

Protein-engineering of Hydroperoxidases

Overview

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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We investigate the effects of exchanging residues lining the lowest part of the main substrate channel in catalase A from bakers yeast (SCC-A). In this protein the channel is about 26 Å long, having a large entrance of 17Å diameter which closes to 4.5 Å in its most narrow part. At that point substrates have to move around the strictly conserved Val111 before getting in contact with the haem and the essential distal histidine (Figure 3). The neck formed by Val111 also prevents formation of a continuous solvent phase connecting the distal haem pocket with the surface. This arrangement is thought to restrict passage to molecules of less than 3.5 Å van der Waals diameter, and to ensure passage of asymmetric substrate molecules (e.g. organic hydroperoxides) in the correct orientation. V111 was exchanged by smaller residues (apolar V111A, polar V111S), as well as by a polar side chain of roughly the same size (V111T), and in some cases these modifications were combined with further exchanges in the close neighbourhood of residue 111. It was shown by X-ray crystallography that additional water molecules are positioned at the “neck” level of the channel in the mutant V111A, closing the gap in the solvent chain characteristic for the wild type. According to modelling, the same should be also true for V111S and V111T. Surprisingly, the reaction rates of V111T both with hydrogen peroxide alone, and with H2O2 + aliphatic alcohols, nearly matched those of the wild type enzyme. V111S showed decreased catalytic activity in both types of reaction, whereas V111A revealed decreased catalatic activity (30%), but up to 200-fold increased capability to oxidize medium chain aliphatic alcohols. Both mutant enzymes showed, however, rather high reactivity towards aromatic 1-electron donors (i.e. they effectively catalysed a classical peroxidase reaction). Interestingly all mutants, as well as most double mutants derived from them showed higher conformational stability than the wild type. We conclude from these rather intriguing results that the channel neck not necessarily has to be apolar to favour the entrance of hydrogen peroxide against other potential reactants, nor is a gap in the chain of solvent molecules necessary to maintain high rates of substrate passage. We suggest that the peroxidatic activity of the mutants is due to the escape of reactive oxygen intermediates (most likely OH-radicals) from the active site, which would not occur in the wild type. This view is supported by the results from steady state and stopped-flow kinetic experiments with peracetic acid as substrate. Currently we try to provide direct experimental evidence for this hypothesis.

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We suggested alternative models for the post-translational formation of the unique pseudoknot in catalase (schematic representation of one example in Figure 4), which we test with catalase A from Saccharomyces cerevisiae. The basic idea of our experimental outset is to “freeze” intermediates of this assembly process. To allow this, cysteine residues were introduced by site-directed mutagenesis in different zones of potential contact between subunits. When pairs of such residues come into close contact, covalent disulfide bridges would form and such stabilise (“freeze”) the respective transient interaction.
A large number of single residue exchanges were performed to introduce cysteines at 4 different regions in the protein monomer. Unfortunately, the unavoidable side-effects of these modifications were too large in one of these regions, whereas in each of the other 3 regions we obtained at least one pair of engineered cysteines which would interact in the planned manner, without reflecting serious side-effects (the pairs C115/C115, C114/C115 and C115/C252 between the b-barrels of the R-related dimer; the pair C45/C349 between the N-terminal arm and the b-barrel of the Q-related dimer; and the pair C26/C127 between the N-terminal arm and the wrapping loop of the R-related dimer). However, under normal conditions of catalase expression, the rate of disulfide formation turned out to be too low. This could not be significantly improved by increased oxidative stress, nor by heterologous expression in the methanotrophic yeast Pichia pastoris. So finally we direct catalase A through the secretory pathway by fusion with the respective signal peptide. This includes passage through the ER which strongly favours formation of disulfide bonds, and to a sufficient extent leads to secretion of the correctly folded and assembled protein into the medium. Due to these experimental problems, which were not expected to this extent, we only very recently started with the experiments which should reveal the pathway of the in vivo assembly of the enzyme; this includes truncation of the N-terminal arm, truncation of the C-terminal a-helical domain with or without parts of the wrapping domain and major modifications of the loop part of the wrapping domain. Each of these truncations will prevent one distinct type of inter-subunit interaction found in the native fold. The pattern of disulfide-linked intermediates observed in each case should then allow to deduce the in vivo-order of domain interaction and subunit assembly. So far only preliminary results are available.

The more general implications of this project could be the (to our knowledge for the first time) successful formation of stable, covalent bonds between the subunits of a soluble protein species. In addition to their potential to solve the problem of the formation of the pseudoknot, these modifications also dramatically increase the stability of the enzyme (catalases show irreversible dissociation upon dilution; since this is accompanied by complete loss of activity, applications of catalases in biotechnology or pharmacology up to now are very limited).

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The site-directed replacement of L47 (and, to a lesser degree, also L46), a residue of the N-terminal arm close to the turn where it bends back in an “elbow” like structure, by cysteine lead to an intrinsically unstable protein, which slowly rearranged to a stable dimeric species with still tightly bound haem. The electronic haem spectrum, virtually identical to that of cytochrome P450cam (CYP101), indicates a thiolate ligand in the fifth coordination position. Obviously a major rerarrangement of the entire proximal haem surrounding occurs, helix a9 being replaced by the N-terminal arm, the newly introduced cysteine residue occupying the position of the former proximal haem ligand Y355 (see Figures 5 and 6 for a schematic model). This spontaneous refolding is enhanced by external perturbation of the C-terminal part of the protein, and by non-ionic detergents at low ionic strength. It no longer functions as catalase, but has increased peroxidatic activity. It also reflects haloperoxidase, as well as monooxygenase (N-demethylation of N.N-dimethylaniline) activity. Bound NADPH obviously acts as ultimate electron donor. So this mutant may act as a single-polypeptide mixed-function oxidase, and could serve as a simple, stable model for the P450 family of major pharmaceutical interest. We will soon start with introducing cysteines in helix a9 to generate similar protein species based on the original catalase fold.

Theses

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Publications

1. Ruis H., and Koller F. (1997). Biochemistry, molecular biology, and cell biology of yeast and fungal catalases. in Oxidative stress and molecular biology of antioxidant defenses. (Scandalios,J.G., ed.), CSH Lab. Press, Cold Spring Harbor, New York, pp. 309-342.
   
   
2. Berthet S., Nykyri L.M., Bravo J., Mate M.J., Berthet-Colominas C., Alzari P.M., Koller F., and Fita I. Crystallization and structural analysis of catalase A from Saccharomyces cerevisiae. Protein Sci. (1997) 6, 481-483.
   
   
3. Zamocky M., Janecek S., and Koller F. (1997). The area of the main substrate channel is highly conserved among all true catalases. Biologia 52, 723-730.
   
   
4. Volf I., Koller E., Bielek E., Koller F. (1997). Colocalization of gold-labelled LDL and fibrinogen on human platelets: enhanced fibrinogen binding induced by LDL. Am. J. Physiol. 273, C118-C129.
   
   
5. Janecek S., Zamocky M., Koller F. (1998). Indication of a common ancestry for copper tyrosinases and heme catalases revealed by hydrophobic cluster analysis of the brown locus protein sequence. Protein Eng. 11, 501-504.
   
   
6. Zamocky M., Koller F. (1999). Understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis. Progr. Biophys. Mol. Biol. 72, 19-66.
   
   
7. Mate M.J., Zamocky M., Nykyri L. M., Herzog C., Alzari P. M., Betzel C., Koller F., Fita I. (1999). Structure of catalase A from Saccharomyces cerevisiae. J. Mol. Biol. 286, 135-149.
   
   
8. 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.
   
   
9. Zamocky M., Janecek S., and Koller F. (2000). Common phylogeny of catalase-peroxidases and ascorbate peroxidases. Gene 256, 169-182.
   
   
10. 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.
    
    
11. Li Sh., Peck-Radosavljevic M., Koller E., Koller F., Kaserer K., Kreil A., Kapiotis S., Hamwi A., Weich H. A., Valent P., Angelberger 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
    

Collaborations

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