J. Mol. Biol. (1991) 221, 35-37
Crystallization and Preliminary X-ray Data of Bromoperoxidase from Streptomyces aureofaciens ATCC
H. Sobekl, T. Haag2, 0. Heifer’, D. Schomburg’, F. Lingens2 and K.-H. van P&x’? 1Department of Molecular Structure Research GBF-Gesellscha~ fiir Biotechnologische Forschung Mascheroder We9 1, D-3300 Braunschweig, Germany 21n.stitute of Microbiology, University of Hohenheim Garbenatr. 30, D-7000 Stuttgart .?‘O,Germany (Received 20 March 1991; accepted 1 May 1991) Bromoperoxidase from Streptornyces aurwfaciem ATCC 10762, a non-haem haloperoxidase, has been crystallized using the hanging drop method. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group P2,3 with a = 1234 A. The asymmetric unit contains a dimer of M, = 60,200. The crystals diffract to at least 23 A resolution and are suitable for crystallographic structure analysis. Keywords:
Halogenating enzymes have been isolated from various sources, e.g. fungi (Morris & Hager, 1966; Liu et al., 1987), marine algae (Manthey & Hager, 1981; Vilter, 1984), bacteria (van Pee & Lingens, 1985; van Pee et al, 1987), white blood cells (Dumontet & Rousset, 1983) and milk (Olson & Little, 1983). All enzymes isolated so far, which catalyse the halogenation of organic compounds, are haloperoxidases. The substrates of these haloperoxidases are peroxides (e.g. hydrogen peroxide), halide ions (chloride, bromide and iodide) and organic halogen acceptors. According to their prosthetic group or cofactor, three groups of haloperoxidases are distinguished. The first group consists of the well studied haem haloperoxidases (Dawson & Sono, 1987). The second group contains vanadium instead of haem (De Boer et al., 1986). No prosthetic group or metal is detectable in bacterial non-haem haloperoxidases (van Pee et al., 1987; Wiesner et al., 1988; Krenn et al., 1988), which represent the third group. So far nothing is known about the reaction bacterial non-haem mechanism of these haloperoxidases. Canine myeloperoxidase, which belongs to the first group, has been crystallized (Fenna, 1987). Another haloperoxidase, a vanadium-dependent bromoperoxidase from Ascophyllum nodosum, has
-also been crystallized (Miiller-Fahrnow et al., 1988). From the third group of haloperoxidases no crystallization has been reported until now. The bromoperoxidase from Streptomyces aurwATCC 10762 (M. Weng, 0. Pfeifer, facien.3 S. Krauss, F. Lingens & K.-H. van Pee, unpublished results) belongs to the third group of halopergene has -been oxidases. The bromoperoxidase cloned and expressed in S. lividum TK 64. High yields of the cloned enzyme were obtained by the following isolation procedure: heat treatment at 75 “C, adsorption chromatography on calciumtartrate and gel filtration on Sephacryl S 300 (M. Weng, 0. Pfeifer, S. Krauss, F. Lingens & K.-H. van PQ, unpublished results). In order to get information about the tertiary structure of an enzyme that catalyses the oxidation of bromide ions without involvement of a prosthetic group or cofactor, we crystallized this bacterial nonhaem bromoperoxidase. Crystallization was performed at room temperature using the hanging drop method. Samples (5 ~1) containing 10 mg protein/ml (in 5 mv-ammonium acetate buffer (pH 68)) were mixed with 5 ~1 of 1.5 to 1.8 M-ammonium sulphate in 50 mM-Tris . HCl buffer (pH 80), and were equilibrated against the same buffer. Crystals of pyramidal shape appeared within a week and grew up to 63 mm edge length. Precession photographs were taken with a Huber precession camera mounted on a Rigaku RU-200 rotating anode X-ray generator at 50 kV and 100 mA using graphite monochromated CuKa
t Correspondence concerning crystallography should be sent to H. Sobek; correspondence concerning the biochemistry of bromoperoxidaae should be sent to K.-H. van PBe. 0022-2836/91/17003&03
0 1991 Academic Press Limited
Figure 1. h/c0 precession
of a crystal
radiation. Precession photographs (Fig. 1) showed that the crystals belong to the cubic space group P2,3 with a = 123.4 A (1 A = 0.1 nm). Assuming a dimeric enzyrne of molecular mass 60,200 Da in the asymmetric unit a crystal packing parameter (Matthews, 1968) of V,,, = 2.6 a3/dalton was calculated. The crystals diffract to at least 2.3 A resolution. A native data set has been collected using a Xentronic area detector and a search for heavyatom derivative data is in progress. We thank S. Weissflog for skilful technical assistance and Drs B. Hofmann and H.-J. Hecht for helpful discussions. This work was supported by a Heisenberg grant’ to K.-H. v.P. and by the Bundesministerium fiir Forschung und Technologie under the contract number 03197164.
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Edited by R. Huber