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Development and Optimazation of Fatty Acids Hydroxylation using Hybrid P450 Enzymes
1. A new approach to fatty acid hydroxylation using hybrid P450-BM3 enzyme and light Ngoc Huynh Lionel Cheruzel* Department of Chemistry San José State University The 23rd Annual Meeting of the Northern California Undergraduate Research Symposium 05/14/11
2. Introduction Cytochromes P450: Belong to a unique superfamilyheme-thiolate enzymes. Catalyze the oxidation of organic molecules including the synthesis of steroids and detoxification of metabolites. Play important roles in catametabolism. C–H C–OH P450-BM3: Hydroxylate long chain fatty acids (C12-C16) selectively at omega positions Easy to handle 2e-, O2, 2H+ P450
3. Mechanism Scheme 1. Catalytic pathway of P450 and the altenate peroxide shunt in generation of Compound I. Ener, M.; Lee, Y.; Winkler, J; Gray, H; Cheruzel, L. Photooxidation of cytochrome P450-BM3PNAS, 2010, 107, 18783-18786
5. Method Protein Expression: Plasmid containing double mutants C62A and C156G of the P450-BM3 heme domain in vector pCWori+ QuikChange Site-Directed Mutagenesis Kit: generate K97C and Q397C mutants Mutants are expressed with a His-tag in E. coli BL21(DE3) cells and purified by size-exclusion and ion exchange columns. Ener, M.; Lee, Y.; Winkler, J; Gray, H; Cheruzel, L. Photooxidation of cytochrome P450-BM3PNAS, 2010, 107, 18783-18786
6. Method Ru(II)-diimine synthesis and labeling reaction: 5:1 label:protein Labeled a) DCC, ethyl acetate; b) 5-aminoPhen, CH3CN; c) Ru(bpy)2Cl2, EtOH/water; d) DTT, Tris buffer
12. Heat at 80-85°C for 20mins5 mins Cirino, P.; Arnold, F. Adv. Synth. Catal. 2002, 344, 932-937 Regioselectivity and Activity of Cytochrome P450 BM-3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide
13. Product detection GC/MS: Mass spectrometer: Bombards molecules by high-energy e- and converts them to ions, which are accelerated in an electric field. Accelerated ions with different mass-to-charge ratio are separated in a magnetic or electric field. These are detected by a device that can count the number of ions striking it.
27. Product Formation under different systems WT + 10mM H2O2 Ru-Q397C-BM3 + 100mM DTC w3 IS w2 w1
28. Product Formation under different systems WT + 10mM H2O2 Ru-Q397C-BM3 + 100mM DTC Ru-Q397C-BM3 + 100mM DTC Ru-Q397C-BM3 + 100mM DTC + catalase catalase + 100mM DTC w3 IS w2 w1
29. Product Formation under different systems WT + 10mM H2O2 Ru-Q397C-BM3 + 100mM DTC Ru-Q397C-BM3 + 100mM DTC Ru-Q397C-BM3 + 100mM DTC + catalase catalase + 100mM DTC w3 IS w2 w1 TTN of Ru-Q397C-BM3 in the presence of catalase increases to 50 times greater
30. Conclusion Successfully engineered first generation of hybrid enzyme P450-BM3 and optimized reaction condition. The activity is enhanced 50 times more than the current systems. Our data suggests the mechanism involves in generating H2O2 locally.
31. Future directions Generate 2nd generation of the hybrid enzyme to take up various substrates. Utilize spectrophotochemistry to investigate the mechanism and kinetics of the hybrid enzymes.
34. Protein lab grant SJSU PROTEIN Lab, Grant# 0923573 (MRI) for the use of the newly-acquired ESI-QTOF/LC MS/MS mass spectrometer.Phuong Huynh Hai Au Mary Copper
