X-ray crystal structures of active site mutants of the vanadium-containingchloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, he terologously expressed in Saccharomyces cerevisiae, have been determined bo th in its apo and in its hole forms at 1.66 and 2.11 Angstrom resolution, r espectively. The crystal structures reveal that the overall structure of th is enzyme remains nearly unaltered, particularly at the metal binding site, At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interact ions and hydrogen bonds with positively charged residues involved in the in teractions with the vanadate in the native protein. The vanadate binding po cket seems to form a very rigid frame stabilising oxyanion binding. The rig idity of this active site matrix is the result of a large number of hydroge n bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine o f the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mu tations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involv ed in forming a salt bridge with Arg490 which also interacts with the vanad ate, The enzymatic chlorinating activity is drastically reduced to approxim ately 1% in mutants D292A, H404A and H496A. The structures of the mutants c onfirm the view of the active site of this chloroperoxidase as a rigid matr ix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by repla cement of large side chains by alanines is usually occupied by a new solven t molecule which partially replaces the hydrogen bonding interactions to th e vanadate. The new solvent molecules additionally replace part of the inte ractions the mutated side chains were making to other residues lining the a ctive site frame. When this is not possible, another side chain in the prox imity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.