AN INVESTIGATION OF HYOSCYAMUS NIGER HYOSCYAMINE 6β-HYDROXYLASE ACTIVITY AND IMPROVEMENT OF EXPRESSION METHODS

Open Access
Author:
Han, Yilin
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • J. Martin Bollinger, Thesis Supervisor
  • David Gilmour , Honors Advisor
Keywords:
  • Biochemistry
  • Bioinorganic
  • H6H
  • Enzyme
Abstract:
Hyoscyamine 6β-hydroxylase from Hyoscyamus niger (Hn H6H) is an iron- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenase that converts hyoscyamine to scopolamine in a two-step reaction via 6-hydroxyhyoscyamine. The first step of this reaction involves a hydroxylation at C6 of hyoscyamine with a similar mechanism deployed by many Fe/2OG hydroxylasess: a classical pathway that reacts the enzyme cofactor with O2 to generate a high-spin Fe(IV)-oxo (ferryl) center, succinate, and CO2. The ferryl abstracts a hydrogen atom (H•) from C6, and radical coupling of the resultant OH ligand with the substrate radical yields the hydroxylated product and regenerates the Fe(II) form of the cofactor. The second step of the reaction is the formation of the epoxide ring between C6 and C7 of the substrate. The exact mechanism is not known yet and is the focus of the current study. Our goal of dissecting the chemical mechanism of Hn H6H by the tools developed in the Bollinger/Krebs group will demand a large quantity of pure protein. In this research project, several methods were used to improve the expression and purification of Hn H6H. Fractional precipitation with ammonium sulfate on wt Hn H6H was not successful due to the tendency of the protein to precipitate across a broad range of ammonium sulfate concentrations. Appendage of a C-terminal hexa-histidine tag by recombinant DNA methods proved to be unsuccessful due to the lack of expression after transformation with the over expression vector. A C-terminal strep-tactin tag was attempted, but successful transformation of the expression strain of E. coli with the plasmid construct was not acheived. A vector expressing a His6-SUMO tagged Hn H6H was assembled and used to transform the over-expression strain. It successfully directed expression of Hn H6H in E. coli BL21DE3 cells. The purification of the tagged enzyme enabled the isolation of a pure version of the enzyme. However, an examination of its activity through LC-MS showed it to be inactive, possibly due to a residual N-terminal extension of the protein left behind after proteolytic removal of the affinity tag.