Ngoc HuynhLionel Cheruzel*

Department of ChemistrySan José State University

The 23rd Annual Meeting of the Northern California Undergraduate Research Symposium

05/14/11

Introduction Cytochromes P450:

Belong to a unique superfamily heme-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

HO

O

2e-, O2, 2H+

HO

O OH

P450

2e-, O2, 2H+

P450

Mechanism

Ener, M.; Lee, Y.; Winkler, J; Gray, H; Cheruzel, L. Photooxidation of cytochrome P450-BM3 PNAS, 2010, 107, 18783-18786

Scheme 1. Catalytic pathway of P450 and the altenate peroxide shunt in generation of Compound I.

Hybrid P450-BM3 enzyme

O

HO HO

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-BM3 PNAS, 2010, 107, 18783-18786

Labeled

Method Ru(II)-diimine synthesis and labeling reaction:

5:1 label:protein

OHI

O

OI

O

I

O

RuN

N

HN I

O

N

N

HN

OI

SH

RuN

N

HN

OS

22

a) b) c) d)

a) DCC, ethyl acetate; b) 5-aminoPhen, CH3CN; c) Ru(bpy)2Cl2, EtOH/water; d) DTT, Tris buffer

Characterization of labeled proteinUV-Vis SpectrumFluorescence SpectrumMass Spectrum

Hybrid P450-BM3 enzyme

O

HO HO

Experiment Setup34.3 M WT

1.5mM lauric acid

5 mins

300 L of 100mM H2O2 (10mM)

600 L of 100mM Tris, pH 8.38

5 mins

300 L of 100mM H2O2 (10mM)• Extraction 2x with CH2Cl2

• Silylation:- 1:1 pyridine:BSTFA+1%TMCS-Heat at 80-85°C for 20mins

2.7mL

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

Product detectionGC/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.

HO

O OH

O

O OSi

MW = 360g/mol

pyridine:BSTFA

1:1

Si

ω1

345

345

117

117

73

131

73

331

ω2

ω3

131 331

73

145

317

145145

317

IS

ω1ω2ω3

WT “Peroxide Shunt” Pathway

IS = O

O

S iO

S i

Hybrid P450-BM3 enzyme

HOHO

O

HO HO

Proposed Photocatalytic Cycle

RuII

RuII*

RuI

O2

DDox

D = diethyldithiocarbamate (DTC)

hν

Experiment Setup

10 M Ru-Q397C-BM3

1.5mM lauric acid

100mM DTC

100mM Tris, pH 8.38

2mL

SilylationExtraction

Product formation under different systems

3.093 4424506 3.093 4781780 7696005 3.093 4443241 5557543 3.093 5262889

3.098 4418506 3.098 4813454 7746983 3.098 4535940 5673489 3.098 5266643

3.104 4423205 3.104 4809980 7741392 3.104 4493206 5620038 3.104 5385764

3.11 4439376 3.11 4839960 7789643 3.11 4503216 5632559 3.11 5403773

3.115 4457051 3.115 4838915 7787961 3.116 4588739 5739530 3.115 5390071

3.121 4465121 3.121 4847603 7801944 3.121 4610046 5766180 3.121 5294037

3.127 4498447 3.127 4864161 7828593 3.127 4624404 5784139 3.127 5363130

3.133 4516887 3.133 4907659 7898600 3.133 4633907 5796025 3.133 5488377

3.138 4530144 3.138 4909695 7901877 3.138 4658188 5826395 3.138 5490391

3.144 4547935 3.144 4936730 7945389 3.144 4671144 5842601 3.144 5514210

3.15 4565294 3.15 4971448 8001265 3.15 4716633 5899498 3.15 5559219

3.155 4574161 3.155 4986540 8025555 3.156 4748198 5938979 3.155 5573670

3.161 4631072 3.161 5007715 8059635 3.161 4750069 5941319 3.161 5577551

3.167 4591060 3.167 5026421 8089741 3.167 4788574 5989480 3.167 5602940

3.173 4647396 3.173 5034834 8103281 3.173 4845488 6060668 3.173 5663885

3.178 4688364 3.178 5072745 8164297 3.178 4845511 6060696 3.178 5664616

3.184 4744013 3.184 5076383 8170152 3.184 4873309 6095466 3.184 5715618

3.19 4769270 3.19 5075903 8169380 3.19 4853694 6070932 3.19 5761565

3.196 4794411 3.196 5104778 8215852 3.196 4906579 6137079 3.196 5818385

3.201 4807207 3.201 5151343 8290796 3.201 4944903 6185014 3.201 5870798

3.207 4848324 3.207 5150768 8289871 3.207 4963616 6208420 3.207 5889980

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

15.4 15.9 16.4 16.9

Ab

un

dan

ce

Retention time (min)

WT+10mM H2O2

Ru-Q397C-BM3+100mM DTC

3

2 1

IS

WT + 10mM H2O2

Ru-Q397C-BM3 + 100mM DTC

Other experiments

H2O2 DTC Light Product

WT

+ - -

- + - X

- + + X

WT/Ru(II)-dimine

+ - -

- + - X

- + + X

Ru-Q397C-BM3

+ - -

- + - X

- - + X

- + +

Kinetic studies

0.1mM LA 0.5mM LA 0.8mM LA 1.0mM LA 1.5mM LA

Kinetic studies

Table 1. The Michelis-Menten kinetic parameters for different reaction systems

Km kcat TTN

WT + H2O2 1.71E-3 0.29 ~ 2

Ru-Q397C-BM3 + H2O2 0.31E-3 0.42 ~ 3

Ru-Q397C-BM3 + DTC + light 0.28E-3 0.56 ~ 30

Experiment Setup

Reaction Mixture: 3uM Ru-Q397C-BM3 1.5mM lauric acid 100mM DTC 12mg catalase

SilylationExtraction

Product Formation under different systems

3

21

IS

WT + 10mM H2O2

Ru-Q397C-BM3 + 100mM DTC

Product Formation under different systems

3

21

IS

WT + 10mM H2O2

Ru-Q397C-BM3 + 100mM DTC

Ru-Q397C-BM3 + 100mM DTCRu-Q397C-BM3 + 100mM DTC + catalase

catalase + 100mM DTC

Product Formation under different systems

3

21

IS

WT + 10mM H2O2

Ru-Q397C-BM3 + 100mM DTC

Ru-Q397C-BM3 + 100mM DTCRu-Q397C-BM3 + 100mM DTC + catalase

catalase + 100mM DTC

TTN of Ru-Q397C-BM3 in the presence of catalase increases to 50 times greater

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.

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.

HO

O

O NO2

12-p-nitrophenol lauric acid

Acknowledgement

Phuong HuynhHai AuMary Copper

• NIH SC3 GM095415 for financial support• SJSU/CSU research mini-grants for financial support.• Protein lab grant SJSU PROTEIN Lab, Grant# 0923573

(MRI) for the use of the newly-acquired ESI-QTOF/LCMS/MS mass spectrometer.

Prof. Lionel Cheruzel

Ngoc-Han TranYen Nguyen Garrett Chavez

Jeremiah Heredia Angelina Nguyen Thuba Bui