Peroxide-driven cytochrome P450 oxygenase variants
AssigneeCalifornia Institute of Technology
InventorsPatrick C. Cirino, Frances H. Arnold
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Abstract
The invention relates to novel variants of cytochrome P450 oxygenases. These variants have an improved ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. These variants also have an improved thermostability as compared to the cytochrome P450 BM-3 F87A mutant. Preferred variants include cytochrome P450 BM-3 heme domain mutants having I58V, F87A, H100R, F107L, A135S, M145A/V, N239H, S274T, L324I, I366V, K434E, E442K, and/or V446I amino acid substitutions.
Claims41
An isolated nucleic acid encoding a variant of a parent cytochrome P450 oxygenase comprising the amino acid sequence of SEQ ID NO:13, wherein the variant consists of a first mutation at amino acid residue F87 of SEQ ID NO:13 and at least one second mutation selected from the group consisting of K9, I58, E93, H100, F107, K113, A135, M145, N186, D217, M237, N239, S274, L324, I366, K434, E442, and V446, of SEQ ID NO:13 and wherein the variant has an enhanced capacity of using peroxide as an oxygen donor compared to the parent.
An isolated nucleic acid encoding a variant of a wild-type parent Bacillus megaterium monooxygenase-3 (BM-3) comprising the amino acid sequence of SEQ ID NO:2, wherein the variant consist of mutations in heme domain amino acid sequences of BM-3 and wherein the variant heme domain amino acid sequences consist of a first mutation at residue F87 of SEQ ID NO:13 and one or more second mutations selected from the group consisting of K9, I58, E93, H100, F107, K113, A135, M145, N186, D217, M237, N239, S274, L324, I366, K434, E442, and V446 of SEQ ID NO:13, wherein the variant has an enhanced capacity of using peroxide as an oxygen donor compared to the parent.
An isolated nucleic acid encoding a variant of a wild-type parent Bacillus megaterium monooxygenase-3 (BM-3) enzyme comprising the amino acid sequence of SEQ ID NO:2, wherein the variant consists of mutations in heme domain amino acid sequences, and wherein the variant has an enhanced capacity of using peroxide as an oxygen donor compared to the parent, wherein the variant heme domain amino acid sequences consist of a first mutation at residue F87 of SEQ ID NO:13, and one or more second mutations selected from the group consisting of H100, M145, M237, S274, and K434 of SEQ ID NO:13.
An isolated nucleic acid encoding a variant of an isolated heme domain of a wild-type parent Bacillus megaterium monooxygenase-3 (BM-3) cytochrome P450 enzyme comprising the amino acid sequence of SEQ ID NO:13, wherein the variant heme domain consists of a first mutation at amino acid F87 of SEQ ID NO:13, and one or more second mutations selected from the group consisting of K9, I58, E93, H100, F107, K113, A135, M145, N186, D217, M237, N239, S274, L324, I366, K434, E442, and V446 of SEQ ID NO:13.
An isolated nucleic acid encoding a variant of a wild-type cytochrome P450 Bacillus megaterium monooxygenase-3 (BM-3) heme domain comprising the amino acid sequence of SEQ ID NO:13, wherein the variant has an enhanced capacity of using peroxide as an oxygen donor compared to the parent, and wherein the variant consists of a first mutation corresponding to amino acid residue F87 of SEQ ID NO:13 and one or more second mutations at an amino acid residue selected from H100, M145, M237, S274, and K434 of SEQ ID NO:13.
Dependencies are inferred from the claim text.