Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: a rational approach.
The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25°C. The activity constants (k(cat)) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25° C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70° C. The rate of catalysis at temperatures above ~ 70° C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.
Behera
Rabindra Kumar
RK
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Goyal
Sandeep
S
Mazumdar
Shyamalava
S
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002711:Chloride Peroxidase / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism; D004795:Enzyme Stability; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology*
D003573:Cytochrome b Group; D006420:Hemeproteins; D005973:Glutamine; C020730:cytochrome b566; D006418:Heme; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002711:Chloride Peroxidase