Characterization of Enzymes Involved in Butane Characterization of Enzymes Involved in Butane Metabolism from the Pollutant Degrading Metabolism from the Pollutant Degrading

bacterium, bacterium, Pseudomonas butanovoraPseudomonas butanovora

John StenbergJohn Stenberg

Mentor: Dan Arp, Ph.D.Mentor: Dan Arp, Ph.D.

September 1, 2004September 1, 2004

BioremediationBioremediation

As the world population and the demands of agriculture and As the world population and the demands of agriculture and industry increase, the availability of fresh water continues to industry increase, the availability of fresh water continues to decreasedecrease

The problems associated with depleted or polluted water affect The problems associated with depleted or polluted water affect not only humans, but the plant and animal populations we not only humans, but the plant and animal populations we depend upondepend upon

The solution?The solution?

BioremediationBioremediation: The process by which living organisms act to : The process by which living organisms act to degrade hazardous organic contaminants or transform degrade hazardous organic contaminants or transform hazardous inorganic contaminants to environmentally safe hazardous inorganic contaminants to environmentally safe levels in soils, subsurface materials, water, sludges, and levels in soils, subsurface materials, water, sludges, and residues. residues.

CometabolismCometabolism Definition: the transformation of a non-growth-supporting substrate by a Definition: the transformation of a non-growth-supporting substrate by a

microorganismmicroorganism

Pseudomonas butanovoraPseudomonas butanovora contains a multi-component monooxygenase that is able to catalyze the contains a multi-component monooxygenase that is able to catalyze the degradation of many substrates including trichloroethylene, dichloroethylenes, aromatic structures, degradation of many substrates including trichloroethylene, dichloroethylenes, aromatic structures, and othersand others

Such compounds are not only environmental pollutants, but in many cases, are very stableSuch 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 Once oxidized by a monooxygenase, it is much easier for these compounds to be further degraded

H ClC C

Cl Cl

Trichloroethylene (TCE)

H O ClC C

Cl Cl

TCE epoxide

Ex. Trichloroethylene oxidation

Pseudomonas butanovoraPseudomonas butanovora

Isolated in Japan from activated sludge near an oil Isolated in Japan from activated sludge near an oil refinery refinery

Capable of growth with butane via the oxidation of Capable of growth with butane via the oxidation of butane to 1-butanol as the first step in the terminal butane to 1-butanol as the first step in the terminal oxidation pathwayoxidation pathway

CC44HH1010 + O + O22 CC44HH99OH + HOH + H22OO Also capable of growth with other alkanes (C2–C9), Also capable of growth with other alkanes (C2–C9),

alcohols (C2–C4) and organic acids as sources of alcohols (C2–C4) and organic acids as sources of carbon and energy carbon and energy

Growth on alkanes catalyzed by a soluble butane Growth on alkanes catalyzed by a soluble butane monooxygenase (sBMO)monooxygenase (sBMO)

Butane Monooxygenase(sBMO)

Butane

Terminal Oxidation Pathway of Terminal Oxidation Pathway of Pseudomonas Pseudomonas butanovorabutanovora

Example: butane to butyric acid (further metabolized as fatty acid)Example: butane to butyric acid (further metabolized as fatty acid)

CH3

CH2

CH2

CH3

CH3

CH2

CH2

CH

O

CH3

CH2

CH2

CH2

OH

1-Butanol

Alcohol Dehydrogenases

CH3

CH2

CH2

O

OH

ButyraldehydeButyric Acid

Aldehyde Dehydrogenases

Butane monooxygenaseButane monooxygenase

Responsible for oxidation of butaneResponsible for oxidation of butane CC44HH1010 + O + O22 CC44HH99OH + HOH + H22OO

Three part enzymeThree part enzyme1. Hydroxylase component (BMOH)1. Hydroxylase component (BMOH)

- - contains the substrate binding di-iron active site and is contains the substrate binding di-iron active site and is responsible for the oxidation of butane to 1-butanol responsible for the oxidation of butane to 1-butanol

2. Reductase component (BMOR)2. Reductase component (BMOR)- responsible for the transfer of electrons from NADH+H- responsible for the transfer of electrons from NADH+H++ to the to the hydroxylase component hydroxylase component

3. Component B (BMOB)3. Component B (BMOB)- coupling protein required for substrate oxidation, electron - coupling protein required for substrate oxidation, electron

transfer ??transfer ??

Proposed Catalytic Cycle of BMOProposed Catalytic Cycle of BMO

Adapted from Wallar, B.J. and J.D. Lipscomb, 1996, Chem. Rev. 96: 2625-2657

BMO Research ObjectivesBMO Research Objectives

Purification and characterization of BMO Purification and characterization of BMO componentscomponents ReductaseReductase HydroxylaseHydroxylase

BMO ActivityBMO Activity Methane oxidationMethane oxidation

Steps leading to PurificationSteps leading to Purification

1. Grow 1. Grow Pseudomonas butanovoraPseudomonas butanovora cells cells Sealed flasks, carboysSealed flasks, carboys Butane 7% overpressureButane 7% overpressure

2. Harvest cells through centrifugation2. Harvest cells through centrifugation 3. Prepare cell-free extract3. Prepare cell-free extract

Lysis by freeze/thaw and sonicationLysis by freeze/thaw and sonication Centrifuge at 46,000 x Centrifuge at 46,000 x g g

Enzyme PurificationEnzyme Purification Multiple column processMultiple column process

1. Q Sepharose resin column 1. Q Sepharose resin column (anion exchange purification)(anion exchange purification)

2. 22. 2ndnd Q Sepharose column Q Sepharose column

3. Gel filtration3. Gel filtration

Superdex 75 – reductaseSuperdex 75 – reductase

Sephacryl S-300 - Sephacryl S-300 - hydroxylasehydroxylase

What so far?What so far?-Purified reductase with activity-Purified reductase with activity

-Partially purified hydroxylase with -Partially purified hydroxylase with activityactivity

Pharmacia FPLC System

sBMO Reductase PurificationsBMO Reductase Purification

97.4

45

66.2

31

21.5

14.4

CFE Q1 Q2 S 75

Purified Reductase FractionsPurified Reductase Fractions

Reductase PropertiesReductase Properties

AA270/458270/458 ratio: 3.1 - 3.7, which is ratio: 3.1 - 3.7, which is similar to the methane similar to the methane monoxygenase reductase monoxygenase reductase and other purified and other purified oxygenase reductasesoxygenase reductases

AA458/340458/340 ratio: 1.4, also similar ratio: 1.4, also similar to the methane to the methane monoxygenase reductasemonoxygenase reductase

UV/Visible Spectra has UV/Visible Spectra has maxima at 272, 340, ~ 400, maxima at 272, 340, ~ 400, 458 nm458 nm

Reductase UV/Visible Spectra

StepStepDCPIP ReductionDCPIP Reduction

(µmol min(µmol min-1-1 mg protein mg protein-1)-1)Fold PurificationFold Purification

Cell Free ExtractCell Free Extract 5.8 ± 0.15.8 ± 0.1 11

Q1Q1 44 ± 0.844 ± 0.8 88

Q2Q2 86 ± 1.586 ± 1.5 1515

Superdex 75Superdex 75 115 ± 1.4115 ± 1.4 2020

Reductase activity and fold purificationReductase activity and fold purification

BMOH

Hydroxylase PurificationHydroxylase Purification

1st Q Sepharose Column Spectra1st Q Sepharose Column Spectra

M Q1 Q2 S-300 S-300

97.4

4566.2

31

21.5

14.4

Hydroxylase Purification StepsHydroxylase Purification Steps

StepStepEO productionEO production

(nmol min(nmol min-1-1 mg protein mg protein-1)-1)% Recovery% Recovery

Whole CellWhole Cell 300300 100100

Cell Free Cell Free ExtractExtract

106106 3535

11stst Q Q Sepharose Sepharose ColumnColumn

231231 7777

BMO Hydroxylase activity during initial BMO Hydroxylase activity during initial purification stepspurification steps

Measured by ethylene oxide (EO) production by gas Measured by ethylene oxide (EO) production by gas chromatographychromatography

Methane OxidationMethane Oxidation Methanol ProductionMethanol Production 5 picomol min5 picomol min-1-1 mg protein mg protein-1-1

0

5000

10000

15000

20000

25000

30000

35000

0 10 20 30 40 50 60 70 80

Time (min)

Peak Area

ProgressProgress

Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g of cell mass/carboy with high BMO activityof cell mass/carboy with high BMO activity

Recoverable BMO hydroxylase activities in cell free extracts Recoverable BMO hydroxylase activities in cell free extracts and initial chromotography steps at high activity comparable and initial chromotography steps at high activity comparable to published sMMO purification strategy of Fox to published sMMO purification strategy of Fox et al.et al. (1989) (1989)

BMO reductase purified to homogeneity with demonstrated BMO reductase purified to homogeneity with demonstrated activity; comparable to the sMMO system reductase in activity activity; comparable to the sMMO system reductase in activity and spectral characteristicsand spectral characteristics

Possible methane oxidationPossible methane oxidation

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute Daniel Arp, Ph.D.Daniel Arp, Ph.D.

Brad Dubbels, Ph.D.Brad Dubbels, Ph.D. Arp LabArp Lab

Kevin Ahern, Ph.D.Kevin Ahern, Ph.D.