Investigating the Role of the Proximal Cysteine Hydrogen Bonding Network and Distal Pocket in Chloroperoxidase

Chloroperoxidase (CPO) is one of the most versatile heme enzyme isolated from the marine fungus, Caldariomyces fumago. Functionally, CPO can catalyze four types of reactions: peroxidation (peroxidase-like), dismutation (catalase-like), halogenation (halogenase-like), and peroxygenation (P450-like). Structurally, CPO has a distal and proximal pockets that can be best described as a hybrid of classical peroxidase and P450s. As a heme-thiolate protein, CPO contains the conserved proximal Pro28-Cys29-Pro30 stretch found in other members of the family. However, the structural and functional roles of these proline residues remain poorly understood. Site-directed mutagenesis was undertaken to generates three CPO mutants, P28A-, P30A-, P28A/P30A-CPO. The replacement of the rigid proline with a more flexible alanine residue, freed up the back bone amide for the formation of additional amide-sulfur hydrogen bond, allowing the investigation of the importance of these residues in CPO catalysis. The three CPO mutants displayed dramatic difference in ligand binding affinity and catalytic activities relative to WT-CPO. Any mutations on the proline resides within the proximal loop eliminated the halogenation and dismutation activities but enhanced the vii epoxidation and peroxidation activities by 4-14 fold. As the binding affinity for cyanide, the CPO mutants displayed significantly higher dissociation constant relative to WT-CPO. Our results revealed that Pro28 and Pro30 play important roles in maintaining the versatility of CPO. As a versatile enzyme, CPO has great application potential in pharmaceutical and chemical industry due to its ability to catalyze the formation of chiral epoxides. Phe103 and Phe186 located on the distal pocket have been proposed to guard the access of substrates to the ferryl oxygen of the heme center. The interactions of these two phenylalanine residues restricted the size of substrates and regulates CPO’s enantioselectivity. F186A- and F103A/F186A-CPO were generated and characterized where the rate of peroxidation and epoxidation were significantly enhanced at the expense of halogenation and dismutation activities. Our results demonstrated that Phe186 played a subtler role relative to Phe103 in terms of substrate specificity and product enantioselectivity of CPO