STRUCTURAL CHARACTERISATION OF ACTIVE SITE MUTANTS OF THE VANADIUM-CONTAINING CHLOROPEROXIDASE FROM THE FUNGUS CURVULARIA INAEQUALIS
S. Macedo-Ribeiro1, W. Hemrika2, R. Renirie2, R. Wever2 and A. Messerschmidt1
1Max-Planck-Institut für
Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18 A,
D-82152 Martinsried, Germany,
2E.C. Slater Institute, Department of
Biochemistry, University of Amsterdam, Plantage Muidergracht 12,
1018 TV Amsterdam, The Netherlands
Keywords: crystal, sulfate-binding,
mutants
The crystal structure of the first vanadium-containing haloperoxidase from the fungus Curvularia inaequalis1 (CPO) and the proposal of a structural-based mechanism2 allowed further understanding of the chemistry of vanadium in biological systems. A stretch of approximately 150 residues, located at the top of the second four-helix bundle forms the vanadium-binding site. The vanadium is coordinated to His496 and the residues Lys353, Arg360, Ser402, Gly403 and Arg490 form hydrogen bonds with the non-protein oxygen's of vanadate. His404 was proposed to function as the acid-base group in catalysis. Residues Arg490 and Asp292 form a strong salt bridge in the proximity of the active site. Residues His404, His496 and Asp292 were mutated to alanines, the protein material was crystallized and the structures were analyzed in correlation with the biochemical and kinetic data obtained for each of these mutants. Superpositions of the refined protein models with the native enzyme revealed that, within the error limits of the electron density maps, the overall backbone structure remains unchanged. The protein seems to form a rigid matrix allowing the formation of a very well defined anion binding site and single-site mutations do not cause drastic changes in the oxyanion-binding site. Additionally, the structure of the recombinant apoenzyme determined at 1.66A resolution shows that this active site frame provided by the protein residues remains mainly unchanged after removal of the active site vanadate. Instead a sulfate ion from the crystallization buffer was found replacing the vanadate (Fig.1).
Fig.1- The active site of apo-CPO displaying a bound sulfate ion. An omit electron density map contoured at 4s highlights the bound sulfate and the active site histidine residue.
Sequence alignment between this vanadium chloroperoxidase and the vanadium-containing bromoperoxidase from Ascophyllum nodosum, an enzyme which is believed to have a similar catalytic mechanism, revealed high similarities in the metal binding site, but very low homology in the remaining regions. Surprisingly those stretches of the sequence are homologous to the ones present in three families of acid phosphatases indicating similarities in the anion-binding active sites of all these enzymes3,4. The parallel structural and chemical features of phosphate and vanadate were recently reinforced by finding similarities in the structure and chemistry of the active sites from vanadium-containing haloperoxidases and some phosphatases. In addition it could be proved that the apochloroperoxidase from the fungus Curvularia inaequalis displayed phosphatase activity4.
Acknowledgments. W.H., R.R and R.W
thank the Netherlands Foundation for Chemical research (SON), the
Netherlands Organization for Scientific Research (NOW) and the
Netherlands Technology Foundation (STW) for financial support. S.
M-R has a fellowship PraxisXXI/BD/4050/94 from Fundaçao para a
Ciencia e Tecnologia (FCT, Portugal).