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

1. Modifications of the Catalytic Properties of Butane Monooxygenase in P. butanovora Sarah Jean Mentors: Dr. Daniel J. Arp Dr. Luis Sayavedra-Soto Kim Halsey

2. Environmental Significance Bioremediation: Use of microorganisms to remove or detoxify toxic or unwanted chemicals from an environment. • Pseudomonas butanovora metabolizes alkanes by oxidizing the alkanes to alcohols • Alkane metabolism importance: • carbon cycling • bioremediation (fossil fuels and chlorinated aliphatic hydrocarbons such as chloroform and trichloroethylene) 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. 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.

3. Introduction to P. butanovora • Uses 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 C4H10 + O2 + AH2 C4H9OH + H2O + A

4. Structural genes of BMO and MMO bmoX bmoY bmoB bmoZ bmoD bmoC hydroxylase, a hydroxylase, b hydroxylase, g reductase mmoX mmoY mmoB mmoZ mmoD mmoC BMO reductase MMO reductase g g a a BUTANOL METHANOL BMO hydroxylase MMO hydroxylase b b BMOB b b MMOB BUTANE a METHANE a g g

5. Approach 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 Y Y A Y WT BMOB mutant BMOB

6. Then What? • Test the effect of the BMOB mutations on sBMO in vitro and in vivo in P. butanova BMO reductase BMO reductase g g a a BMO hydroxylase BMO hydroxylase METHANOL b b ? WT BMOB Mutant BMOB b b a g METHANE a g METHANE

7. Procedure of purification Clone into pET-15b, propagate in DH5-E. Coli Protein Quantification dialysis Plasmid Prep Purified BMOB His-Bind purification Transfer into BL21 E. Coli Add IPTG & grow for 3 hours Grow to OD600 0.6

8. DNA plasmid preparation Sequencing Carry out the mutagenesis reaction DH5a plasmid preps Transformation DH5a Determine concentration of plasmid

9. Methods: BMOB Expressions IPTG Transformation into BL21 Expression to gel His-Bind purification column LB media SDS-PAGE Purified protein After dialysis

10. BMOB Purification Progress

11. Summary • 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

12. Acknowledgements Howard Hughes Medical Institute Dr. Daniel J. Arp Dr. Luis Sayavedra-Soto Kim Halsey Dr. Kevin Ahern Everyone else in the Nitrogen Fixation Lab Thank you for all the help!!