Spatial organization of catalase proteins

Transcription

1 Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 1 & 2, August 1985, pp Printed in India. Spatial organization of catalase proteins B. K. VAINSHTEIN, W. R. MELIK-ADAMYAN, V. V. BARYNIN, A. A. VAGIN and A. I. GREBENKO Institute of Crystallography, Academy of Sciences of the USSR, Moscow , USSR Abstract. The three-dimensional structure of the heme-containing fungal catalase from Penicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2 0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct X-ray primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains a β-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near the β-barrel. A comparison of the structure of catalase from Penicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteria Thermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2 1 3, a = Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme in Thermus thermophilus catalase, the active site is between the four helices and contains two manganese ions. Keywords. Catalase; fungal; organisation; spatial catalase; protein structure; structure catalase; X-ray crystallography. Introduction Catalase (H 2 O 2 :H 2 O 2 oxidoreductase, EC ) is an enzyme responsible for decomposition of hydrogen peroxide to water and molecular oxygen. 2H 2 O 2 2H 2 O + O 2. This enzyme is present in cells of almost all the living organisms, i.e. bacteria, fungi, plants and animals. Nearly all the catalases investigated so far by a number of methods (biochemical studies, sedimentation, electron microscopy and X-ray diffraction) were found to be tetrameric proteins of molecular mass 2,50,000 3,00,000. Each subunit is a single polypeptide chain to which the heme group is bound, being directly involved in the enzymatic process. The catalytic activity of the catalases is very high and is estimated as sec 1 per catalytic site. Properties of catalases are thoroughly reviewed by Deisseroth and Dounce (1970) and by Schonbaum and Chance (1976). Many crystalline forms of catalase were extensively studied by electron microscopy. Abbreviations used: PVC, Penicillium vitale catalase; BLC, beef liver catalase; TTC, Thermus thermophilus catalase. 471

2 472 Vainshtein et al. A remarkable crystalline structure of beef liver catalase was obtained in the form of tubes with monomolecular walls. Structure analysis of these tubes by the method of three-dimensional reconstruction made it possible to establish how molecules are packed, their size and overall shape, and the tetrahedral symmetry 222 of subunit packing (Vainshtein et al., 1968; Gurskaya, 1975; Barynin et. al., 1979) (figure la). Figure 1. The molecular dimensions and quaternary structure of (a) BLC from electron microscopic data (b) BLC from X-ray data (c) PVC from X-ray data. Recently the detailed structural data have been obtained for two heme-containing catalases studied by X-ray crystallography. One is a fungal catalase isolated from Penicillium vitale (PVC) and the other is a beef liver catalase (BLC) (figure 1b and c). We describe here also the structure of a non-heme catalase from bacteria Thermus thermophilus HB8 (TTC). The structure of catalase from Penicillium vitale The PVC crystals belong to the space group P3 1 21, a = Å and c = Å. There are three protein molecules per unit cell, so an asymmetric unit contains half a molecule. The molecular mass of PVC is 2,90,000. X-ray intensities for native protein crystals and heavy-atom derivatives were collected in a four-circle diffractometer up to 3 Å resolution. An electron density map at 6 Å resolution made it possible to determine the molecular dimensions of PVC (figure 1c) and to reveal a 222 symmetry. The 2q axis coincides with the crystallographic two fold axis inherent in the space group, whereas 2p and 2r axes are noncrystallographic, their position being deduced from direct inspection of molecular model (Vainshtein et al., 1979). At the next stages of work, at 3 5 and 3 0 Å resolution the non-crystallographic symmetry were taken into account by electron density averaging in two subunits related by this type of symmetry, with subsequent phase refinement. This procedure led to a

3 Spatial organization of catalase proteins 473 highly improved electron density map which allowed finally to create an atomic model of the molecule, with all backbone atoms along the polypeptide chain and some bulky side chains (Vainshtein et al., 1980, 1981a). The experimental data set was further extended to 2 0 Å resolution using X-ray photographs obtained with a synchrotronous radiation source in the European Molecular Biology Laboratory, Hamburg. These data along with the previous atomic model were used for refinement of the protein structure at 2 Å resolution. Inspection of a difference map calculated with coefficients (2 F O F C ) exp(iφ C ), where F O and F C are observed and calculated structure amplitudes and φ C are calculated phases made it possible to complete the atomic model by an addition of more side chains, with some local rebuilding of the main chain. Since the amino acid sequence of PVC is unavailable, an accurate analysis of the electron density map was made to select for each residue a side chain which gives the best fit to the electron density, with occasional references to the known sequence of BLC. By such a procedure, the X-ray sequence of PVC was derived containing about 500 amino acid residues other than alanine of the total number of 670 residues. The complete atomic model was further refined using the Hendrickson-Konnert program (Hendrickson and Konnert, 1981). The refinement was carried out against 68,000 structure factors in the region between 5 and 2 Å resolution using 5000 atoms of the subunit model. After 24 cycles of refinement, the crystallographic R-factor decreased to The positions of secondary structure elements in the PVC subunit are schematically shown in figure 2. The polypeptide chain is composed of 670 amino acid residues. The first 56 residues, situated away from the subunit globule are involved in many contacts with amino acid residues of neighbouring subunits. In the globular part of a subunit the polypeptide chain forms three domains. The largest domain I consists of 300 residues. It contains a β-barrel of 8 anti-parallel strands, along with 7 helices located near the barrel. After the last helix, the polypeptide chain is running as an extended segment of 70 residues wrapping the large domain and connecting it with the next, smaller domain II, composed, essentially, of 4 helices. This domain contains about 70 residues. The C-terminal domain III is of α/β type. It contains about 150 amino acid residues forming a sheet of 5 parallel β strands and 4 α helices situated above and below the β- sheet. The topology of β strands and helices is similar to that of flavodoxin, although the spatial homology is hardly seen. The 4 PVC subunits are held together by hydrophobic and ionic interactions and hydrogen bonds. An unusual intersubunit contact is observed for subunits related by the p-axis: 13 residues of the N-terminal segment are drawn together through a loop formed by residues of the neighbouring subunit in the wrapping region of the polypeptide chain. The heme group is situated in the region of the large domain deeply inside the tetramer, with the iron atom at a distance of 17 Å from the surface of the molecule. The distal side where hydrogen peroxide is bound was identified from X-ray study of the complex of PVC with a specific inhibitor, aminotriazole. The electron density distribution in the heme region and the derived atomic positions are shown in figure 3. On the proximal side a close contact with the iron atom is made by a tyrosine residue whose phenolic group occupies the fifth coordination position. The plane of the

4 474 Vainshtein et al. Figure 2. Three-dimensional structure of the PVC subunit. The scheme for the arrangement of secondary structure elements. The cylinders correspond to helical segments of the chain, and arrows to extended segments forming the β structure. The two-fold axes of the molecule are indicated as 2p, 2q, 2r aromatic ring is inclined to the porphyrin plane making an angle of 40. On the distal side, nearest to the iron atom is a histidine residue. A small isolated region of positive electron density which is seen in figure 3 close to the iron atom may be interpreted as a water molecule bound at the site otherwise occupied by hydrogen peroxide. Analysis of a space filling function revealed inside the molecule, the presence of rather large cavities with a volume from Å 3. One of these cavities has a connection with the exterior and forms a channel running up to a region very near the heme. One may suggest that this cavity plays an important functional role providing an access to the active site for a substrate molecule.

5 Spatial organization of catalase proteins 475 Figure 3. (a) The electron density distribution and (b) the amino acid residues in the heme region. A comparison of the structures of ΡVC and BLC A comparison of the tertiary structures of PVC (Vainshtein et al., 1981b) and BLC (Murthy et al., 1981) turned out to be very interesting. A conformation of the polypeptide chain in the β-barrel and around the active site was found to be very similar, whereas the N-terminal and C-terminal parts are markedly different.

6 476 Vainshtein et al. The most remarkable is the presence of domain III in the C-terminal part of the ΡVC subunit, which is completely absent in BLC (figure 1b and 1c). Another marked difference is observed in the N-terminal part where in BLC there is an additional segment of 12 residues forming an α-helix. In the remaining parts, i.e. in the first two domains and in the wrapping connecting segment the chain conformation is almost the same, neglecting some small insertions and deletions. Out of the total number of 478 residues in similar parts of the two catalases one may establish an equivalence in position for 458 residues. After superposing these parts, the r.m.s. difference in α-carbon positions was found to be only 1 17 Å. Such a difference is typical for highly homologous proteins. A homology of amino acid sequence in these parts of the chain based on the X-ray sequence of ΡVC is about 35% (175 identical residues). Thus two catalases isolated from organisms widely diverged for many million years in the course of the biological evolution proved to have a very similar structure of domains I and II, but only PVC has domain III. The structure of TTC It would be interesting to select for study another catalase of some other species. For this purpose a catalase from the extremal thermophillic bacteria Thermus thermophilius (strain HB8) has been chosen. This catalase is distinguished from other known catalases by its unique thermostability. It shows 100% activity up to 90 C. The molecular mass of TTC is 2,10,000. Spectroscopic studies revealed an absorption band near 450 nm with a shoulder at 500 nm suggesting the presence of Mn 3+ ion in the molecule. The presence of manganese ions was confirmed further, by electron spin resonance spectra. The molecular mass of a single subunit, based on the electrophoresis data, is 33,000 37,000. Hence the molecule should be composed of six subunits. Electronic spectra from solutions of this catalase showed that the protein does not contain the heme group. All these lines of evidence allow one to suggest a similarity of TTC with Mn-catalase isolated from Lactobacillus plantarum (Kono and Fridovich, 1983). We have succeeded in growing crystals of TTC suitable for X-ray studies. The crystals were extremely stable against X-ray radiation. Their life time under X-ray beam was 2 5 times that of PVC. The crystals are cubic, the space group is P2 1 3, a = Å. The molecule is situated on the 3-fold axis. There are 4 molecules per unit cell, with 1/3 of molecule in asymmetric unit. The structure determination of TTC has been carried out by the method of multiple isomorphous replacement taking into account a non-crystallographic symmetry (table 1). X-ray intensities were collected on the KARD-3 diffractometer with a positionsensitive area detector, developed at the Institute of Crystallography for high-speed experiments with protein crystals (Andrianova et al., 1982). About 1 5 million reflections were measured from crystals of native protein and its heavy-atom derivatives, with 12 equivalents per each unique reflection. The molecular boundaries were determined using an electron density map at 6 Å resolution. The molecule may be approximated as a distorted hexagonal prism with a height of 60 Å and cross-section dimension of 85 Å (figure 4).

7 Spatial organization of catalase proteins 477 Table 1. Data collection and phase refinement parameters for TTC. where Fp and Fph are the observed structure factors for the crystals of the native protein and of the heavy atom derivative respectively, Dph is the calculated structure factor derivative, Ε is the r.m.s. lack of closure error, and fh is the r.m.s. calculated structure factor for the heavy atoms alone. The overall figure of merit at 3 0 Å resolution before and after two cycles of noncrystallographic symmetry phase refinement were M iso = 0 53, M 1 = 0 83, M 2 = Figure 4. Schematical representation of the quaternary structure of TTC molecule and arrangement of the molecules in the unit cell. On this map 8 straight segments of high electron density were clearly seen in an asymmetric unit. They were interpreted as, α helices. From positions of these helices a non-crystallographic two-fold axis has. been identified which relates two protein subunits in the asymmetric part of the unit cell. The point group of the molecule is 32.

8 478 Vainshtein et al. Figure 5. Three-dimensional structure of the TTC subunit: (a) The α-carbon backbone of the TTC subunit and (b) its schematical drawing. An electron density map at 3 Å resolution made it possible to trace the polypeptide chain in the TTC subunit and to localize the active site of the enzyme occupied by two metal ions (figure 5). The subunit skeleton shows up as a bundle of 4 nearly parallel long helices, 2 of them having a length of about 40 Å. At the middle part of the space between the helices a region of high electron density looks like a double peak which is readily conceived as a double site of Mn ions in the active site. The distance between the ions is 3 6 Å. The packing of central helices allows one to include TTC into a family of 4-helical proteins which also contains hemerythrin, cytochromes c' and b 562, apoferritin and the coat protein of tobacco mosaic virus (Weber and Salemme, 1980). TTC is very similar to apoferritin in the packing of 4 long helices with an irregular segment at the middle part of one of them, whereas the hemerythrin subunit shows a similar double metal site between helices formed by two Fe ions. However, this similarity does not extend to the entire subunit because the full length of the polypeptide chain in TTC is twice that in apoferritin and three times of that in hemerythrin. Conclusion Thus, it can be concluded that catalases with a common function are presented in nature as two completely different proteins both in their chemistry and structure, without even a sign of convergence of their active sites. On the other hand, it is

9 Spatial organization of catalase proteins 479 interesting to note that functionally related oxygen-carrying proteins also have their representatives both among heme containing proteins (haemoglobin) and the fourhelical ones bearing a double metal site (hemerythrin). Acknowledgements Authors thank Dr. Yu. V. Nekrasov and Dr. A. N. Popov for help in diffractometer data collection, Dr. S. Hangulov for E.S.R. study of TTC and Dr. V. V. Borisov for helpful discussions. Authors are pleased to acknowledge a contribution of Dr. K. S. Bartels (EMBL, Hamburg, FRG) in collecting of synchrotronous X-ray data on PVC and Prof. M. G. Rossmann and Dr. I. Fita (Purdue University, West Lafayette, USA) in crystallographic refinement of PVC. References Andrianova, M. E., Kheiker, D. M, Popov, A. N., Simonov, V. I., Anisimov, Yu. S., Chernenko, S. P., Ivanov, A. B., Movchan, S. Α., Peshekhonov, V. D. and Zanevsky, Yu. V. (1982) J. Appl. Cryst., 15, 626. Barynin, V. V., Vainshtein, B. K., Zograf, O. N. and Karpukhina, S. Ya. (1979) Mol. Biol. (Moscow), 13, Deisseroth, A. and Dounce, A. L. Physiol. Rev., 50, 319. Gurskaya, G. V. (1975) Kristallographiya, 20, 516. Hendrickson, W. A. and Konnert, J. (1981) in Biomolecular structure, function, conformation and evolution (ed. R. Srinivasan) (Oxford: Pergamon Press) Vol. 1, p. 43. Kono, Y. and Fridovich, I. (1983) J. Biol. Chem., 258, Murthy, M. R. N., Reid, T. J., Sicignano, Α., Tanaka, N. and Rossmann, Μ. G. (1981) J. Mol. Biol., 152, 465. Schonbaum, G. R. and Chance, B. (1976) in The Enzymes (3rd edn., ed. P. D. Boyer) (New York: Academic Press) Vol. 13, p Vainshtein, Β. Κ., Barynin, V. V. and Gyrskaya, G. V. (1968) Dokl. Akad. Ν auk USSR, 182, 569. Vainshtein, Β. Κ., Melik-Adamyan, W. R., Barynin, V. V., Vagin, Α. Α., Nekrasov, Yu. V., Malinina, L. V., Gulyi, M. F., Gudkova, L. V. and Degtyar, R. G. (1979) Dokl. Akad. Nauk USSR, 246, 220. Vainshtein, Β. Κ., Melik-Adamyan, W. R., Barynin, V. V. and Vagin, Α. Α. (1980) Dokl. Akad. Nauk USSR, 250, 242. Vainshtein, Β. Κ., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A. and Grebenko, A. I. (1981a) Nature (London), 293, 411. Vainshtein, Β. Κ., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A. and Grebenko, A. I. (1981b) Kristallografia, 26, Weber, P. C. and Salemme, F. R. (1980) Nature (London), 287, 82.