Cytochrome P450 BM3 enzyme variants for preparation of cyclopropanes
The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.
1. A P450 BM3 enzyme variant that can cyclopropanate an olefinic substrate, wherein the P450 BM3 enzyme variant comprises an iron heme, an amino acid sequence having at least 95% identity to SEQ ID NO:1, and at least one amino acid substitution selected from the group consisting of an Ala substitution at position 78, a Val substitution at position 87, a Ser substitution at position 142, an Ile substitution at position 175, a Val substitution at position 184, an Arg substitution at position 226, a Gln substitution at position 236, a Gly substitution at position 252, an Ala substitution at position 268, a Val substitution at position 290, a Val substitution at position 353, a Val substitution at position 366, and a Lys substitution at position 442.
2. The P450 BM3 enzyme variant of claim 1 , further comprising a substitution mutation at the axial position of the heme coordination site at position 400.
3. The P450 BM3 enzyme variant of claim 2 , wherein the mutation is a substitution of Cys with Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position.
4. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant comprises the amino acid sequence set forth in SEQ ID NO:1 and one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following amino acid substitutions: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K.
5. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant comprises one, two, or all three amino acid substitutions selected from the group consisting of an Ala substitution at position 263, a Gly substitution at position 328, or a substitution at position 438.
6. The P450 BM3 enzyme variant of claim 5 , wherein the substitution at position 438 is an Ala substitution, a Ser substitution, or a Pro substitution.
7. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant comprises from one to five active site alanine substitutions at positions 75, 177, 181, 263, or 437.
8. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant comprises an Ala substitution at position 268 and a substitution at position 400, wherein the substitution at position 400 is any amino acid other than Cys.
9. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant has a higher total turnover number (TTN) compared to the wild-type sequence.
10. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant produces a plurality of cyclopropanation products having a Z:E ratio of from 1:99 to 99:1.
11. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant produces a plurality of cyclopropanation products having at least 30% to at least 90% diasteroselectivity.
12. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant produces a plurality of cyclopropanation products having at least 30% to at least 90% enantioselectivity.
13. The P450 BM3 enzyme variant of claim 1 , wherein the P450 BM3 enzyme variant is in lyophilized form.
14. A cell expressing a P450 BM3 enzyme variant of claim 1 .
15. An expression vector comprising a nucleic acid sequence encoding a P450 BM3 enzyme variant of claim 1 .
16. A cell comprising the expression vector of claim 15 .