Presentation Title

Mutations of Cysteine Residues in Lysyl Oxidase

Faculty Mentor

Karlo Lopez

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

50

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Lysyl oxidase is copper-dependent amine oxidase that catalyzes the oxidative deamination of lysine residues in collagen and elastin. Lysyl oxidase is held together by five disulfides and a crosslinked catalytic cofactor. This project aims to investigate the role each disulfide plays in the stability and function of the enzyme. Each cysteine residue will be mutated to a serine, with the C238S mutant having been confirmed by DNA sequencing. Mutated enzyme was overexpressed in an E. coli system and 15.3 mg of enzyme was purified. Activity was tested using a coupled peroxidase/amplex red assay and the enzyme was shown to have a specific activity of 0.01 U/mg. This is consistent with literature values but much lower than wildtype activity shown in recombinant lysyl oxidase. The enzyme also showed a 3% copper incorporation suggesting that the mutation has an effect on the active-site structure. The data was obtained using 6 M urea in 50 mM Tris buffer, pH 7.6, to solubilize the enzyme. For consistency the values reported will be verified using a recently published overexpression system that does not require urea. This requires that the mutated enzyme be tagged with NUS-A to increase solubility and each mutant will be used as template to mutate the next cysteine until all 10 cysteines have been changed to serine. This would allow us to better understand the role of each cysteine in the enzyme.

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Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

Mutations of Cysteine Residues in Lysyl Oxidase

50

Lysyl oxidase is copper-dependent amine oxidase that catalyzes the oxidative deamination of lysine residues in collagen and elastin. Lysyl oxidase is held together by five disulfides and a crosslinked catalytic cofactor. This project aims to investigate the role each disulfide plays in the stability and function of the enzyme. Each cysteine residue will be mutated to a serine, with the C238S mutant having been confirmed by DNA sequencing. Mutated enzyme was overexpressed in an E. coli system and 15.3 mg of enzyme was purified. Activity was tested using a coupled peroxidase/amplex red assay and the enzyme was shown to have a specific activity of 0.01 U/mg. This is consistent with literature values but much lower than wildtype activity shown in recombinant lysyl oxidase. The enzyme also showed a 3% copper incorporation suggesting that the mutation has an effect on the active-site structure. The data was obtained using 6 M urea in 50 mM Tris buffer, pH 7.6, to solubilize the enzyme. For consistency the values reported will be verified using a recently published overexpression system that does not require urea. This requires that the mutated enzyme be tagged with NUS-A to increase solubility and each mutant will be used as template to mutate the next cysteine until all 10 cysteines have been changed to serine. This would allow us to better understand the role of each cysteine in the enzyme.