Peroxidase activity;
Rational protein design;
Therm us thermophilus;
CYP175A1;
Heme active site;
Site directed mutagenesis;
RESONANCE RAMAN-SPECTROSCOPY;
BETA-CAROTENE HYDROXYLASE;
HORSERADISH-PEROXIDASE;
THERMUS-THERMOPHILUS;
HYDROGEN-PEROXIDE;
SUBSTRATE HYDROXYLATION;
CATALYTIC HISTIDINE;
CATALASE-PEROXIDASE;
CRYSTAL-STRUCTURE;
IRON LIGAND;
D O I:
10.1016/j.jinorgbio.2010.07.008
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
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 Glu 183 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 degrees 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 degrees C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was -70 degrees C. The rate of catalysis at temperatures above -70 degrees 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 similar to 5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins. (C) 2010 Elsevier Inc. All rights reserved.
机构:Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Auclair, K
Moënne-Loccoz, P
论文数: 0引用数: 0
h-index: 0
机构:Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Moënne-Loccoz, P
de Montellano, PRO
论文数: 0引用数: 0
h-index: 0
机构:
Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USAUniv Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
机构:
DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756
BERRY, EA
TRUMPOWER, BL
论文数: 0引用数: 0
h-index: 0
机构:
DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756
机构:Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Auclair, K
Moënne-Loccoz, P
论文数: 0引用数: 0
h-index: 0
机构:Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Moënne-Loccoz, P
de Montellano, PRO
论文数: 0引用数: 0
h-index: 0
机构:
Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USAUniv Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
机构:
DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756
BERRY, EA
TRUMPOWER, BL
论文数: 0引用数: 0
h-index: 0
机构:
DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756DARTMOUTH COLL,HITCHCOCK MED CTR,DARTMOUTH MED SCH,DEPT BIOCHEM,HANOVER,NH 03756