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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!!