STRUCTURAL INVESTIGATIONS ON COFACTOR-FREE HALOPEROXIDASES. H. J. Hecht1, B. Hofmann1, J. Altenbucher2, K. H. van Pée3, 1GBF (Gesellschaft für Biotechnologische Forschung),Dept. MSF, Mascheroder Weg 1, D-38124 Braunschweig, FRG, 2Univ. Stuttgart, Inst. Indust. Genetik, D-70569 Stuttgart, FRG, 3TU Dresden, Inst. Biochemie, D-01062 Dresden, FRG
Haloperoxidases catalyse the halogenation of organic compounds in the presence of halide ions and peroxides like H2O2. The bacterial haloperoxidases catalyse this redox reaction without the involvement of a known cofactor1,2. In order to elucidate the reaction mechanism recently the crystal structure of the cofactor-independent haloperoxidase CPO-A2 from the 7-chloro-tetracycline- producing Streptomyces aureofaciens ATCC 10762 was solved by MIR3. The overall structure of this haloperoxidase has been characterized as a [[alpha]]/[[beta]]-hydrolase fold with a catalytic triad in the active center consisting of Ser 98, His 257 and Asp 228, but the exact mechanism of the enzyme remained still unclear. The halogenation reaction of these haloperoxidases requires the presence of organic acids like acetate or propionate. Therefore as reaction mechanism it was postulated, that acetate, activated by the nucleophile Ser O[[gamma]], could be oxidised by peroxide to the peroxoacid in a first step, which then could oxidize the halide ion, probably with the involvement of as yet unidentified additional residues in the active center.
Crystals of haloperoxidase CPO-A2 were obtained at pH 8.0, where the enzyme shows only low residual activity3. As part of this investigation of the reaction mechanism we determined the structures of the related haloperoxidase from Streptomyces lividans (CPO-L) both at pH 8.0 and at pH 6.0, the activity pH optimum. The role of M99 in CPO-A2 was investigated with the CPO-A2 mutant Met99-->Thr which is analogous in this position to CPO-L but inactive. The structure of this mutant has also been determined and refined at 1.5 Å. We present here these three structures and discuss their differences and the implications for the reaction mechanism.
CPO-A2 M99T from Streptomyces aureofaciens crystallizes from 1.8 M ammonium sulphate at pH 8.0. Crystals diffract to 1.5 Å resolution, belong to spacegroup I23 with a = 121.7 Å and contain one monomer in the asymmetric unit. CPO- L from Streptomyces lividans TK64 crystallizes at pH 8.0 from 2.1 M ammonium sulphate in the space group I4 with a = 176.5 Å, c = 64.0 Å. Crystals diffract to 1.9 Å and contain one trimer per asymmetric unit. At pH 6.0 CPO-L crystals grow from 2.0 M ammonium sulphate in space group P212121 and diffract to 2.6 Å.The structures have been solved by molecular replacement using AMORE and have been refined using X-PLOR.
1. van Pée, K.-H. et al. Biol. Chem. Hoppe-Seyler 368, 1225-1232 (1987)
2. Wiesner, W. et al. J. Biol. Chem. 263, 13725-13732 (1988)
3. Hecht et al., Nat. Struct. Biol. 1, 532-537 (1994)