IP Library › Granted Patent US 9,145,571

Granted Patent B2

US 9,145,571 · App. 13/391,111 · Granted Sep 29, 2015

Preparation method of human metabolites of simvastatin or lovastatin using bacterial cytochrome P450 and composition therefor

Inventors: Chul Ho Yun (Daejeon, KR); Keon Hee Kim (Daejeon, KR); Dong Hyun Kim (Chungcheongnam-Do, KR); Ji Yeon Kang (Daejeon, KR); Sun Ha Park (Gwangju, KR)

Assignee: Chul Ho Yun

C12P17/06 C12N9/0071 C12Y114/14001

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Quick Facts

Patent No.: US 9,145,571
App. No.: 13/391,111
Granted: Sep 29, 2015
Kind: B2

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Abstract

A method of producing human metabolites of simvastatin or lovastatin, and more particularly, a method of producing human metabolites of simvastatin and lovastatin by using bacterial cytochrome P450 BM3(CYP102A1) or mutants thereof, and a composition and kit therefor.

Claims (160)

1. A composition for catalyzing a reaction for preparing human metabolites of simvastatin or lovastatin, the composition comprising at least one mutant of CYP102A1 having a reductase domain and a heme domain, wherein the at least one mutant of CYP102A1 is a polypeptide having the amino acid sequence of SEQ ID NO:16 with substitutions in the reductase domain between amino acid positions 473-1048 and at least one substitution in the heme domain between amino acid positions 1-472, wherein the at least one substitution in the heme domain is selected from the group consisting of:

substituting the arginine at amino acid position 47 with alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), phenylalanine (F) or tryptophan (W);

substituting the tyrosine at amino acid position 51 with A, V, L, I, P, M, F or W; substituting the glutamic acid at amino acid position 64 with glycine (G), serin (S), threonine (T), cysteine (C), tyrosine (Y), asparagine (N) or glutamine (Q);

substituting the alanine at amino acid position 74 with G, S, T, C, Y, N or Q;

substituting the phenylalanine at amino acid position 81 with A, V, L, I, P, M or W;

substituting the leucine at amino acid position 86 with A, V, I, P, M, F or W;

substituting the phenylalanine at amino acid position 87 with A, V, L, I, P, M or W;

substituting the glutamic acid at amino acid position 143 with G, S, T, C, Y, N or Q;

substituting the leucine at amino acid position 188 with G, S, T, C, Y, N or Q;

substituting the alanine at amino acid position 264 with G, S, T, C, Y, N or Q; and

substituting the glutamic acid at amino acid position 267 with A, V, L, I, P, M, F or W; and

wherein the at least one mutant of CYP102A1 further comprises:

substituting the alanine at amino acid position 474 with V, L, I, P, M, F or W;

substituting the glutamic acid at amino acid position 558 with aspartic acid (D);

substituting the threonine at amino acid position 664 with A, V, L, I, P, M, F or W;

substituting the proline at amino acid position 675 with A, V, L, I, M, F or W;

substituting the alanine at amino acid position 678 with glutamic acid (E) or D;

substituting the glutamic acid at amino acid position 687 with A, V, L, I, P, M, F or W;

substituting the alanine at amino acid position 741 with G, S, T, C, Y, N or Q;

substituting the lysine at amino acid position 813 with E or D;

substituting the arginine at amino acid position 825 with G, S, T, C, Y, N or Q;

substituting the arginine at amino acid position 836 with lysine (K) or histidine (H);

substituting the glutamic acid at amino acid position 870 with G, S, T, C, Y, N or Q;

substituting the isoleucine at amino acid position 881 with A, V, L, P, M, F or W;

substituting the glutamic acid at amino acid position 887 with G, S, T, C, Y, N or Q;

substituting the proline at amino acid position 894 with G, S, T, C, Y, N or Q;

substituting the serine at amino acid position 954 with G, T, C, Y, N or Q;

substituting the methionine at amino acid position 967 with A, V, L, I, P, F or W;

substituting the glutamine at amino acid position 981 with R, K or H;

substituting the alanine at amino acid position 1008 with D or E;

substituting the histidine at amino acid position 1021 with G, S, T, C, Y, N or Q; and

substituting the glutamine at amino acid position 1022 with D or E.

2. The composition of claim 1 , wherein the at least one mutant of CYP102A1 comprises one or more substitutions selected from the group consisting of:

R47L/L861/F87V/L188Q/A474V/E558D/T664A/P675L/A678E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#13V2);

R47L/E64G/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#15V3);

R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A 1008D/H1021Y/Q1022E (M#16V2); and

R47L/E64G/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678 E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#17V2).

3. A kit for producing human metabolites of simvastatin or lovastatin, the kit comprising the composition according to claim 1 and a NADPH-generating system.

4. A method of producing human metabolites of simvastatin or lovastatin comprising the steps of reacting the composition of claim 1 with simvastatin or lovastatin.

5. The method of claim 4 , further comprising adding a NADPH-generating system to the reaction.

6. A composition for catalyzing a reaction for preparing human metabolites of simvastatin or lovastatin, the composition comprising at least one mutant of CYP102A1 having a reductase domain and a heme domain, wherein the at least one mutant of CYP102A1 is a polypeptide having the amino acid sequence of SEQ ID NO:16 with substitutions in the reductase domain between amino acid positions 473-1048 and at least one substitution in the heme domain between amino acid positions 1-472, wherein the at least one substitution in the heme domain is selected from the group consisting of:

substituting the arginine at amino acid position 47 with L;

substituting the tyrosine at amino acid position 51 with F;

substituting the glutamic acid at amino acid position 64 with G;

substituting the alanine at amino acid position 74 with G;

substituting the phenylalanine at amino acid position 81 with I;

substituting the leucine at amino acid position 86 with I;

substituting the phenylalanine at amino acid position 87 with A or V;

substituting the glutamic acid at amino acid position 143 with G;

substituting the leucine at amino acid position 188 with Q;

substituting the alanine at amino acid position 264 with G; and

substituting the glutamic acid at amino acid position 267 with V; and

wherein the at least one mutant of CYP102A1 further comprises:

substituting the alanine at amino acid position 474 with V;

substituting the glutamic acid at amino acid position 558 with D;

substituting the threonine at amino acid position 664 with A;

substituting the proline at amino acid position 675 with L;

substituting the alanine at amino acid position 678 with E;

substituting the glutamic acid at amino acid position 687 with A;

substituting the alanine at amino acid position 741 with G;

substituting the lysine at amino acid position 813 with E;

substituting the arginine at amino acid position 825 with S;

substituting the arginine at amino acid position 836 with H;

substituting the glutamic acid at amino acid position 870 with N;

substituting the isoleucine at amino acid position 881 with V;

substituting the glutamic acid at amino acid position 887 with G;

substituting the proline at amino acid position 894 with S;

substituting the serine at amino acid position 954 with N;

substituting the methionine at amino acid position 967 with V;

substituting the glutamine at amino acid position 981 with R;

substituting the alanine at amino acid position 1008 with D;

substituting the histidine at amino acid position 1021 with Y; and

substituting the glutamine at amino acid position 1022 with E.

7. A composition for catalyzing a reaction for preparing human metabolites of simvastatin or lovastatin, the composition comprising at least one mutant of CYP102A1 having a reductase domain and a heme domain, wherein the at least one mutant of CYP102A1 is a polypeptide having the amino acid sequence of SEQ ID NO:16 with substitutions in the reductase domain between amino acid positions 473-1048 and at least one substitution in the heme domain between amino acid positions 1-472, wherein the at least one substitution in the heme domain is selected from the group consisting of:

substituting the arginine at amino acid position 47 with L;

substituting the tyrosine at amino acid position 51 with F;

substituting the glutamic acid at amino acid position 64 with G;

substituting the alanine at amino acid position 74 with G;

substituting the phenylalanine at amino acid position 81 with I;

substituting the leucine at amino acid position 86 with I;

substituting the phenylalanine at amino acid position 87 with A or V;

substituting the glutamic acid at amino acid position 143 with G;

substituting the leucine at amino acid position 188 with Q;

substituting the alanine at amino acid position 264 with G; and

substituting the glutamic acid at amino acid position 267 with V; and

wherein the at least one mutant of CYP102A1 further comprises:

substituting the lysine at amino acid position 473 with T

substituting the alanine at amino acid position 474 with V;

substituting the glutamine at amino acid position 546 with E;

substituting the aspartic acid at amino acid position 599 with E;

substituting the valine at amino acid position 624 with L;

substituting the aspartic acid at amino acid position 637 with E;

substituting the lysine at amino acid position 639 with A;

substituting the glycine at amino acid position 660 with R;

substituting the threonine at amino acid position 664 with A;

substituting the glutamine at amino acid position 674 with K;

substituting the threonine at amino acid position 715 with A;

substituting the alanine at amino acid position 716 with T;

substituting the alanine at amino acid position 741 with G;

substituting the alanine acid at amino acid position 782 with V;

substituting the lysine at amino acid position 813 with E;

substituting the isoleucine at amino acid position 824 with M;

substituting the glutamic acid at amino acid position 870 with N;

substituting the isoleucine at amino acid position 881 with V;

substituting the glutamic acid at amino acid position 887 with G;

substituting the aspartic acid at amino acid position 893 with G;

substituting the glutamic acid at amino acid position 947 with K;

substituting the serine at amino acid position 954 with N;

substituting the methionine at amino acid position 967 with V;

substituting the alanine at amino acid position 1008 with D; and

substituting the aspartic acid at amino acid position 1019 with E.

8. A mutant of CYP102A1, wherein the mutant comprises substitutions selected from the group consisting of:

R47L/L861/F87V/L188Q/A475V/E559D/T665A/P676L/A679E/E688A/A742G/K814E/R826S/R837H/E871N/1882V/E8880/P895S/S955N/M968V/Q982R/A1009D/H1022Y/Q1023E (M#13V2),

R47L/E64G/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#15V3),

R47L/F811/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#16V2),

R47L/E64G/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678 E/E687A/A7410/K813E/R825S/R836H/E870N/1881V/E8870/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E (M#17V2), and

R47L/E64G/F811/F87V/E143G/L188Q/E267V/K474T/A475V/Q547E/D600E/V625 L/D638E/K640A/0661R/T665A/Q675K/T716A/A717T/A7420/A783V/K814E/1825M/E871N/1882V/E8880/D8940/E948K/S955N/M968V/A1009D/D1020E (M#17V8).

9. A composition for catalyzing a reaction for preparing human metabolites of simvastatin or lovastatin, the composition comprising at least one mutant of CYP102A1 having a reductase domain and a heme domain, wherein the at least one mutant of CYP102A1 is a polypeptide having the amino acid sequence of SEQ ID NO:16 with substitutions in the reductase domain between amino acid positions 473-1048 and at least one substitution in the heme domain between amino acid positions 1-472, wherein the at least one substitution in the heme domain is selected from the group consisting of:

substituting the arginine at amino acid position 47 with alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), phenylalanine (F) or tryptophan (W);

substituting the tyrosine at amino acid position 51 with A, V, L, I, P, M, F or W;

substituting the glutamic acid at amino acid position 64 with glycine (G), serin(S), threonine (T), cysteine (C), tyrosine (Y), asparagine (N) or glutamine (Q);