1. Characterization of Enzymes Involved in Butane Metabolism from the Pollutant Degrading bacterium, Pseudomonas butanovora John Stenberg John Stenberg Mentor: Dan Arp, Ph.D. Mentor: Dan Arp, Ph.D. September 1, 2004 September 1, 2004

2. Bioremediation  As the world population and the demands of agriculture and industry increase, the availability of fresh water continues to decrease  The problems associated with depleted or polluted water affect not only humans, but the plant and animal populations we depend upon  The solution?  Bioremediation: The process by which living organisms act to degrade hazardous organic contaminants or transform hazardous inorganic contaminants to environmentally safe levels in soils, subsurface materials, water, sludges, and residues.

3. Cometabolism  Definition: the transformation of a non-growth-supporting substrate by a microorganism  Pseudomonas butanovora contains a multi-component monooxygenase that is able to catalyze the degradation of many substrates including trichloroethylene, dichloroethylenes, aromatic structures, and others  Such compounds are not only environmental pollutants, but in many cases, are very stable  Once oxidized by a monooxygenase, it is much easier for these compounds to be further degraded H Cl C Cl Trichloroethylene (TCE) H O Cl C Cl TCE epoxide Ex. Trichloroethylene oxidation

4. Pseudomonas butanovora  Isolated in Japan from activated sludge near an oil refinery  Capable of growth with butane via the oxidation of butane to 1-butanol as the first step in the terminal oxidation pathway C 4 H 10 + O 2 C 4 H 9 OH + H 2 O  Also capable of growth with other alkanes (C2–C9), alcohols (C2–C4) and organic acids as sources of carbon and energy  Growth on alkanes catalyzed by a soluble butane monooxygenase (sBMO)

5. Butane Monooxygenase (sBMO) Butane Terminal Oxidation Pathway of Pseudomonas butanovora Example: butane to butyric acid (further metabolized as fatty acid) 1-Butanol Alcohol Dehydrogenases Butyraldehyde Butyric Acid Aldehyde Dehydrogenases

6. Butane monooxygenase  Responsible for oxidation of butane C 4 H 10 + O 2 C 4 H 9 OH + H 2 O C 4 H 10 + O 2 C 4 H 9 OH + H 2 O  Three part enzyme 1. Hydroxylase component (BMOH) - contains the substrate binding di-iron active site and is responsible for the oxidation of butane to 1-butanol 2. Reductase component (BMOR) - responsible for the transfer of electrons from NADH+H + to the hydroxylase component 3. Component B (BMOB) 3. Component B (BMOB) - coupling protein required for substrate oxidation, electron transfer ??

7. Proposed Catalytic Cycle of BMO Adapted from Wallar, B.J. and J.D. Lipscomb, 1996, Chem. Rev. 96: 2625-2657

8. BMO Research Objectives  Purification and characterization of BMO components Reductase Reductase Hydroxylase Hydroxylase  BMO Activity Methane oxidation Methane oxidation

9. Steps leading to Purification  1. Grow Pseudomonas butanovora cells Sealed flasks, carboys Sealed flasks, carboys Butane 7% overpressure Butane 7% overpressure  2. Harvest cells through centrifugation  3. Prepare cell-free extract Lysis by freeze/thaw and sonication Lysis by freeze/thaw and sonication Centrifuge at 46,000 x g Centrifuge at 46,000 x g

10. Enzyme Purification  Multiple column process 1. Q Sepharose resin column (anion exchange purification) 2. 2 nd Q Sepharose column 3. Gel filtration Superdex 75 – reductase Sephacryl S-300 - hydroxylase  What so far? -Purified reductase with activity -Partially purified hydroxylase with activity Pharmacia FPLC System

11. sBMO Reductase Purification 97.4 45 66.2 31 21.5 14.4 CFE Q1 Q2 S 75

12. Purified Reductase Fractions Reductase Properties A 270/458 ratio: 3.1 - 3.7, which is similar to the methane monoxygenase reductase and other purified oxygenase reductases A 458/340 ratio: 1.4, also similar to the methane monoxygenase reductase UV/Visible Spectra has maxima at 272, 340, ~ 400, 458 nm Reductase UV/Visible Spectra

13. Step DCPIP Reduction (µmol min -1 mg protein -1) Fold Purification Cell Free Extract 5.8 ± 0.1 1 Q1 44 ± 0.8 8 Q2 86 ± 1.5 15 Superdex 75 115 ± 1.4 20 Reductase activity and fold purification

14. BMOH Hydroxylase Purification 1st Q Sepharose Column Spectra

15. M Q1 Q2S-300 97.4 45 66.2 31 21.5 14.4    Hydroxylase Purification Steps

16. Step EO production (nmol min -1 mg protein -1) % Recovery Whole Cell 300100 Cell Free Extract 10635 1 st Q Sepharose Column 23177 BMO Hydroxylase activity during initial purification steps  Measured by ethylene oxide (EO) production by gas chromatography

17. Methane Oxidation  Methanol Production  5 picomol min -1 mg protein -1

18. Progress  Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g of cell mass/carboy with high BMO activity  Recoverable BMO hydroxylase activities in cell free extracts and initial chromotography steps at high activity comparable to published sMMO purification strategy of Fox et al. (1989)  BMO reductase purified to homogeneity with demonstrated activity; comparable to the sMMO system reductase in activity and spectral characteristics  Possible methane oxidation

19. Acknowledgements  Howard Hughes Medical Institute  Daniel Arp, Ph.D.  Brad Dubbels, Ph.D.  Arp Lab  Kevin Ahern, Ph.D.