Three-dimensional structure of polyketide synthases
View Patent ↗The present invention comprises crystalline polyketide synthases, isolated non-native polyketide synthases having the structural coordinates of said crystalline polyketide synthases, and nucleic acids encoding such non-native polyketide synthases. Also disclosed are methods of predicting the activity and/or substrate specificity of putative polyketide synthase, methods of identifying potential polyketide synthase substrates, and methods of identifying potential polyketide synthase inhibitors.
1. A method for crystallizing the chalcone synthase of SEQ ID NO:1 or mutant thereof,
wherein said mutant chalcone synthase has one or more mutations selected from the group consisting of Xaa 164 S, Xaa 164 D, H 303 A, H 303 Q, H 303 D, H 303 T, N 336 A, N 336 D, N 336 Q, N 336 H, F 215 S, F 215 Y, and F 215 W,
wherein said method comprises growing crystals of said chalcone synthase or mutant thereof by vapor diffusion at 4° C. in 2 μL drops,
wherein said drops contain a 1:1 mixture of 25 mg/mL of said chalcone synthase and crystallization buffer, and
wherein said crystallization buffer comprises 2.2–2.4 M ammonium sulfate and 0.1 M PIPES, pH 6.5.
2. The method of claim 1 further comprising 5 mM DTT.
3. A crystal produced by the method of claim 1 .
4. The crystal of claim 3 where said chalcone synthase is defined by SEQ ID NO:1.
5. The crystal of claim 3 wherein said mutation is Xaa 164 S.
6. The crystal of claim 3 wherein said mutation is Xaa 164 D.
7. The crystal of claim 3 wherein said mutation is H 303 A.
8. The crystal of claim 3 wherein said mutation is H 303 Q.
9. The crystal of claim 3 wherein said mutation is H 303 D.
10. The crystal of claim 3 wherein said mutation is H 303 T.
11. The crystal of claim 3 wherein said mutation is N 336 A.
12. The crystal of claim 3 wherein said mutation is N 336 D.
13. The crystal of claim 3 wherein said mutation is N 336 Q.
14. The crystal of claim 3 wherein said mutation is N 336 H.
15. The crystal of claim 3 wherein said mutation is F 215 S.
16. The crystal of claim 3 wherein said mutation is F 215 Y.
17. The crystal of claim 3 wherein said mutation is F 215 W.
18. A crystalline form of the chalcone synthase of SEQ ID NO:1, wherein said crystalline form has space group P 3 2 2 1, unit cell dimensions 97.54 Å, 97.54 Å, 65.52 Å, and unit cell angles alpha=90°, beta=90°, gamma=120°,
wherein residue Xaa 164 of SEQ ID NO:1 is selected from the group consisting of Cys and Ser.
19. A crystalline complex comprising said crystalline form of chalcone synthase according to claim 18 and a chalcone synthase substrate.
20. The crystalline complex of claim 19 , wherein said substrate is selected from the group consisting of chalcone, naringenin, resveratrol, cerulenin, acyl-CoA, malonyl-CoA, and hexanoyl-CoA.