| Abstract
| - CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure isavailable, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxinreductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. TheT214V mutation increases the rate by facilitating substrate binding and enhancing the associated spinstate change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxinto CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450camcan, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme.This information can be used to engineer by mutagenesis an improved complementarity of the protein−protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As aresult, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalyticsystem that hydroxylates fatty acids.
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