Designing Metabolons for Improving the Efficiency of Enzyme Cascades

Shelley Minteer, PhD Department of Chemistry and Materials Science & Engineering University of Utah Metabolons

• In 1985, Srere first proposed the concept “metabolon” describing that: Sequential enzymes within most metabolic pathways, such as Tricarboxylic Acid (TCA) Cycle enzymes, are physically associated to form structural-functional complexes.

2 Biochem. Soc. Symp. 1987, 54, 173 O

O- -O

OH O

Malate

O

O- -O

O O

Oxaloacetate

Biochemistry 1996, 39, 12652

Substrate channeling Increasing local concentration

Advantages!

Improving enzyme performance Facilitating an efficient flux 3 Previous Work: In-situ Crosslinking

• Cross-linker Application: – Bi-functional conjugation In situ covalent cross-linking reagents with varying of Krebs cycle enzymes within the spacer arm lengths: mitochondria before isolation, using glutaraldehyde or DMS • Dialdehydes • Imidates • Carbodiimides • Succinimides – Only bind surface residues in relatively close proximity – Able to capture and Natural analyze weak or transient metabolon protein interactions 4 J.Am.Chem.Soc. 2010, 132, 6288

Previous Work: Metabolon Biofuel Cell

Enzymes immobilized in Nafion membrane

Biosensors and Bioelectronics 2008, 24, 945 5 J.Am.Chem.Soc. 2010, 132, 6288 Artificial Metabolons

Crowded Chemical Polymer Microenvironment Bonding Entrapment

Concentration Crowding Hydrophobic Cross-linking Immobilization ↑ Reagent Modification

Enzymes being far apart Enzymes in close proximity

Analytical techniques, including: Förster Resonance Energy Transfer

6 Artificial Metabolons

• Formation of metabolons from isolated and purified enzymes

• Looking for attachment sites that are somewhat selective and all for minimization of 1/1 and 2/2 linkages

• Heterobifunctional crosslinking is an obvious choice, but 100 fold decrease in enzyme activity 7 In-vitro Crosslinking

BM(PEG)3

O O

N O N O O O O

S

O N O

Glucose-6-phosphate O O

Dehydrogenase O O

N

S O One CYS O N O

O

O Hexokinase

O

O

N

O S 4 CYS

8 Km (mM) Vmax (nM/min) Sensitivity (mA/mM) Glucose Dehydrogenase 7.48 41.14 10.62 (±1.22) Hexokinase and Glucose-6- 1.07 32.27 69.31 (±7.14) phosphate Dehydrogenase

• Hexokinase/G6PDH electrodes demonstrated the expected lower saturation point exhibited by the lower Vmax • Substrate affinity for the two enzyme system was significantly improved however as demonstrated by the very low Km • This affinity resulted in a 5 fold increase in sensitivity for glucose in the comparison of the linear range of the two glucose biosensors

9 Three types of test electrodes for comparison A B C

Glucose ATP Glucose NAD+ Glucose-6- Glucose Phosphate ATP NADP+ NADPH NADH + NADP+ NADPH 6-phosphogluconate Gluconolactone 6-phosphogluconate

Glucose-6- Bi-enzyme Glucose Phosphate Hexokinase Complex Dehydrogenase Dehydrogenase Schematic representation of the three types of fuel cell electrodes tested: A) Glucose dehydrogenase modified B) Hexokinase and Glucose-6-phosphate dehydrogenase immobilized randomly C) Cross-linked bienzyme complex 10 modified electrodes. Representative Power Curves

20 Control Glucose Dehydrogenase Non-Crosslinked Crosslinked

) 15

2

W/cm

m

10

Power Density ( Density Power 5

0 0 100 200 300 Current Density (mA/cm2) Open Circuit Maximum Maximum Power Potential (V) Current Density Density (mW/cm2) (mA/cm2) Control 0.291 (±0.016) 23.01 (±3.27) 0.531 (±0.102) Glucose 0.410 (±0.003) 69.58 (±24.49) 3.22 (±1.23) Dehydrogenase Two enzyme Non- 0.507 (±0.005) 132.0 (±5.6) 6.73 (±0.60) crosslinked

Bienzyme 0.604 (±0.025) 235.0 (±35.8) 20.60 (±7.68) 11 Complex Analytical Techniques for Evaluating Metabolons

• FRET pair: carboxylic acid, succinimidyl ester – AlexaFluor® 555/647 – React with primary amines at protein surface

Excitation at 545nm

• Repeated emission scan from 560nm to 700nm in Tris buffer (50mM, pH7.4):

Average protein separation:

1/6 r = 5.1nm*[1/(1-Fd-a/Fd )-1] 12 • Protein Separation vs. Concentration

– Three enzymes were equally mixed in Tris buffer (50mM, pH7.4): AlexaFluor® 555-ACO, AlexaFluor® 647-mMDH and CS – Total protein concentration series :

R0

13 Protein Separation vs. Viscosity • Two labeled enzymes: AlexaFluor® 555-ACO and AlexaFluor® 647- mMDH were equally mixed in Tris buffer (50mM, pH7.4) containing crowding reagents at R.T.

R0

14 Enzyme entrapment

– Entrapment in modified-linear polyethylenimine (LPEI)

H Br N N N H 15:1 ACN:methanol H n heated and refluxed Br 0.2 0.8 LPEI C8- LPEI

15 MRS Communications 2011, 1, 37 Immobilization in LPEI

R0

16 Immobilization in Chitosan

R0

17

CS dimer

5.0nm~10.0nm

ACO monomer

ACO monomer

mMDH dimer

18 Biochemistry 1997, 47, 14271 Activity Assays • Enzyme activity assay of malate dehydrogenase O O

- - O mMDH O - - O + NAD+ O + NADH + H+ OH O O O

Malate Oxaloacetate 340nm

Single mMDH mMDH+CS+ACO (unit/mg) (unit/mg)

No polymer,solution 1.35±0.18 1.75±0.06

LPEI,solution 1.69±0.04 1.85±0.07

C8-LPEI,solution 1.27±0.07 2.24±0.07

19 Conclusion

• The in-vitro enzyme association can be enhanced by simulating inner microenvironment in mitochondria : – Keep the enzymes at a high concentration – Introducing significant amount of crowding reagents • Polymer entrapment and encapsulation facilitate enzyme interactions by bringing them together

20 Future work

• Use of in-vivo and in-vitro formed metabolons • Thin and more reproducible layers made by slide coater

21 Electron Transport Chain Metabolon

• Study all individual redox enzymes • Use substrates and inhibitors to study electrochemical actuation

Gold Electrode 22 Not drawn to scale Natural Metabolon Formation

• Metabolon of ETC has been seen in-vivo • Substrate channeling, catalyst density • Has not been electrochemically characterized

10 nm Biochim. Biophys. Acta 2009, 1787, 60–67. J. Bacteriol. 1993, 175, 6377. J. Biol. Chem. 1987, 262, 1786. 23 Isolation and Purification of Complexes

• Mitochondria isolation • Protein chromatography – Ion-exchange, size exclusion, affinity columns • Enzymatic spectroscopic assays • Purification check by Native PAGE • Electrochemical analysis

24 Electrochemistry in Lipid Bilayers

• Gold SAM with 1 mM thiol lipid, overnight soak in ethanol • Bilayer enzyme/lipid mixture with 40 mM sodium deoxycholate • Membrane dialysis to exchange the detergent into lipid bilayers

• Inner membrane composition: Phosphatidylcholine (38%), Cardiolipin (16%), Phosphatidylethanolamine (24%) [Phosphatidylinsitol(16%)]

25 J Bacteriol. 1991, 173, 2026. Ubiquinone: Cytochrome c Reductase

0.1 M phosphate buffer, pH Potential held at 0.3 V 7.4 100 µM cytochrome c injections 50 mV/s 50 µl/min flow rate 26 Complex IV Lipid Membrane Electrode

Thiol lipid

Not drawn to scale • 0.1 M phosphate buffer pH 7.4, degassed

27 Complex III and IV heme enzymes

0.1 M phosphate buffer, pH 7.4 50 mV/s 28 Metabolon Formation

• Can the complexes form a stable metabolon on the electrode? • Study the current produced of the metabolon electrochemistry by the relationship with substrate diffusion and turnover rate of enzymes • How does inhibition affect metabolon activity? • Optimization of enzyme and substrate concentration

Biochemistry 2007, 46, 12579-12585. ChemCatChem 2011, 3, 561-570. Biochemistry 2007, 46, 12579-12585 29 Complex Complex I IV

Complex III

10 nm

30 EMBO Journal 2011, 30, 4652. 30 Possible Configurations

1-Donor All 3 • Two-step FRET • Higher efficiency for long range 2 &3 transfer • Better detection sensitivity of acceptor fluorescence • Multiple samples in the same measurement

Nat. Meth. 2008, 5, 509. 31 JACS 2003, 125, 7336. 31 FRET Pairs 54 • Alexa Fluor 546 – Cytochrome c Reductase R 74 6 o R 71 • Alexa Fluor 594 – Cytochrome c Oxidase Å o 59 Å • Alexa Fluor 647 – NADH UQ:Oxidoreductase 3 Absorption 4 64 Ro 85 Emission 7 Å Alexa Fluor 546 – 556/575 Alexa Fluor 594 – 590/617 Alexa Fluor 647 – 650/665 %

Wavelength/n 32 www.invitrogen.comm Calvin Cycle

• Used by plants to fix CO2 in photosynthesis – Converts short term energy storage from light reactions (ATP, NADH) into sugars and other organic building blocks

• Project goal: Recreate cycle in vitro via bioconjugation of metabolons and optimize to produce sequester carbon dioxide and design responsive bioelectrodes

33

CO2 Capture

34 Methanol Oxidation

NAD+ H COH 3 Alcohol Dehydrogenase

HCOH Electrode Aldehyde Dehydrogenase

HCO2H Formate Dehydrogenase CO2 NADH

A) B) a b Fuels

c d

35 Kim et al Submitted Novel Biosynthetic Pathway for CO2 Fixation Formose Reaction

J. Am. Chem. Soc. 1999, 121:12192-12193. Trends in Biotechnology 2008, 26:375-381. Current Opinion in Biotechnology 2003, 14:454- 459. Nature 2006, 440:940-943. J. Am. Chem. Soc. 1984, 106:482936- 4832. Kinetics and Catalysis 2007, 48:245-254. Tetrahedron Letters 1959, 1:22-26. New Phytologist 1945, 44:17-24.

Design of a formlose enzyme

WT Reaction Target Reaction

WT-BAL with BENZOIN Bound

37 PDB ID: 2AG0 Bioorganic Chemistry 2006, 34:345-361 Design of a Formolase Enzyme

Rosetta Design

Benzaldehyde Lyase Formolase

Benzaldehyde Lyase Formolase -1 -1 Formose Reaction kcat/KM (M s ) 0.05 4.7 -1 -1 Benzoin Reaction kcat/KM (M s ) 1186 >0.02 (n.d.)

Specificity Switch: Substrate Preference of WT/Design >5,000,000

38 Bulk Electrolysis

Modified Toray® Platinum Standard Paper Counter Calomel Electrode Electrode Reference

Applied -0.8 V vs SCE

100 mM 13C Labeled Sodium formate and 1 mM NADH in 100

mM Phosphate Buffer pH 8.0 39

Future work

• Electron transport chain metabolon – Natural – Artificial – Understanding mechanisms for toxin sensing • Calvin cycle metabolon – Natural – Artificial – Electrosynthesis and responsive electrodes • Non-natural metabolons – Electrosynthesis – Biosensing

40