Tuning the substrate specificity by engineering the active site of cytochrome P450cam: a rational approach.
Rational design of the active site of cytochrome P450cam has been carried out to catalyse oxygenation of various potentially important chemical reactions. The modeling studies showed that the distal pocket of the heme consisting of the Y96, T101, F87 and L244 residues could be suitably mutated to change the substrate specificity of the enzyme. We found that the mutant enzymes could catalyse oxygenation of indole to produce indigo. While Y96F was found to be several times better as a catalyst for conversion of indole to indigo, the double mutant Y96F/L244A showed the highest NADH oxidation rate as well as yield of indigo. The oxidative catalysis using H(2)O(2) as the oxygen source was found to produce a higher purity of indigo, and lesser or no formation of indirubin was detected. The enzymatic oxygenation of aromatic hydrocarbons such as coumarin and analogues was also found to be enhanced on mutation of Y96 and L244 residues in the enzyme. The studies also showed that mutation of suitable residues can alter the regio-selectivity of hydroxylation of the aromatic hydrocarbons.
Manna
Soumen Kanti
SK
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
Mazumdar
Shyamalava
S
D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004396:Coloring Agents / Q000138:chemical synthesis / Q000737:chemistry; D005288:Ferredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007203:Indigo Carmine; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
D004396:Coloring Agents; D005288:Ferredoxins; D007211:Indoles; D016877:Oxidants; D009243:NAD; C100137:putidaredoxin; D006861:Hydrogen Peroxide; D007203:Indigo Carmine; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase