Modifications of the Catalytic Properties of Modifications of the Catalytic Properties of Butane Monooxygenase in Butane Monooxygenase in P. butanovoraP. butanovora

Sarah JeanSarah JeanMentors: Dr. Daniel J. ArpMentors: Dr. Daniel J. Arp

Dr. Luis Sayavedra-SotoDr. Luis Sayavedra-SotoKim HalseyKim Halsey

Environmental SignificanceEnvironmental Significance

Pseudomonas butanovora metabolizes alkanes by oxidizing the alkanes to alcohols

Alkane metabolism importance:1. carbon cycling

2. bioremediation (fossil fuels and chlorinated aliphatic hydrocarbons such as chloroform and trichloroethylene)

Bioremediation: Use of microorganisms to remove or detoxify toxic or unwanted chemicals from an environment.

Trichloroethylene: (TCE) A volatile organic compound widely used as an industrial degreaser; as a solvent for oils, paints, and varnishes; and as a dry cleaning agent.

Chloroform: Produced mainly by the chlorination of methane, chloroform is used in the production of refrigerants and agrochemicals. It is no longer used as an anesthetic.

Introduction to Introduction to P. butanovoraP. butanovoraUses a soluble diiron-

containing monooxygenase to metabolize the alkane

Structurally similar to methane monooxygenase (MMO), but can’t metabolize methane

Monooxygenase: introduces one atom of dioxygen into the substrate and reduces the other atom to water

CC44HH1010 + O + O22 + AH + AH22 C C44HH99OH + HOH + H22O + AO + A

Structural genes of BMO and MMOStructural genes of BMO and MMO

bmoX bmoY bmoB bmoZ bmoD bmoC

mmoX mmoY mmoB mmoZ mmoD mmoC

BMO reductase MMO reductase

BMO hydroxylase MMO hydroxylase

BMOBBMOB MMOBMMOB

BUTANE METHANE

BUTANOL METHANOL

hydroxylase, hydroxylase, hydroxylase, hydroxylase, hydroxylase, hydroxylase, reductasereductase

Approach to modify BMOBApproach to modify BMOB

Find out how BMOB determines what alkanes can be metabolized by BMO

Create mutations in the DNA sequence coding BMOB

Purify the modified BMOB

WT WT BMOBBMOB

mutant mutant BMOBBMOB

YY

YYYY

AA

Then What?Then What?

Test the effect of the BMOB mutations on sBMO in vitro and in vivo in P. butanova

BMO reductase BMO reductase

BMO hydroxylase BMO hydroxylase

WT WT BMOBBMOB

Mutant Mutant BMOBBMOB

METHANE METHANE

??

METHANOL

Procedure of purificationProcedure of purification

Clone into pET-Clone into pET-15b, propagate in 15b, propagate in DH5-DH5- E. ColiE. Coli

Plasmid Plasmid PrepPrep

Transfer Transfer into BL21 into BL21 E. E. ColiColi

Grow to Grow to ODOD600600 0.6 0.6

Add IPTG & Add IPTG & grow for 3 grow for 3 hourshours

His-Bind His-Bind purificationpurification

dialysisdialysis

Protein Protein QuantificationQuantification

DNA plasmid preparationDNA plasmid preparation

Carry out the Carry out the mutagenesis mutagenesis

reactionreaction

Transformation DH5Transformation DH5

SequencingSequencing

Determine Determine concentration of concentration of

plasmidplasmid

DH5DH5 plasmid preps plasmid preps

Methods: BMOB ExpressionsMethods: BMOB Expressions

Transformation into Transformation into BL21BL21

IPTGIPTG

LB mediaLB media

Expression to gelExpression to gel His-Bind purification columnHis-Bind purification column

SDS-PAGESDS-PAGE

Purified proteinPurified protein

After dialysisAfter dialysis

BMOB Purification ProgressBMOB Purification ProgressMutants of Mutants of BMOBBMOB

BMOB BMOB cloned into cloned into pET15BpET15B

pET15B pET15B transformed transformed into BL21into BL21

Expression Expression of BMOBof BMOB

Purification Purification of BMOBof BMOB

Dialysis of Dialysis of BMOBBMOB

Protein Protein quantificationquantification

T109AT109A

T108ST108S

N105GN105G

P32HP32H

T108AT108A

T109YT109Y

H8AH8A

M114AM114A

Y100 frame Y100 frame shiftshift

V110AV110A

SummarySummary

8 BMOB mutations were purified and protein concentrations were determined for all of them

Future research: Growth curves and activity assays will be carried out to see if changes in specificity were achieved

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute

Dr. Daniel J. ArpDr. Daniel J. Arp

Dr. Luis Sayavedra-SotoDr. Luis Sayavedra-Soto

Kim HalseyKim Halsey

Dr. Kevin AhernDr. Kevin Ahern

Everyone else in the Nitrogen Fixation LabEveryone else in the Nitrogen Fixation Lab

Thank you for all the help!!Thank you for all the help!!