Abstract
Lysyl oxidase (LOX) is a copper-dependent amine oxidase enzyme that catalyzes the formation of crosslinkages of collagen and elastin in connective tissues by oxidative deamination of lysine. Using site-directed mutagenesis, Histidine 303 has been shown to be a key residue that acts as the necessary catalytic base for this enzyme to function properly. Histidine 303 was mutated to isoleucine to remove catalytic activity and to aspartate and glutamate, respectively, in order to provide alternate residues that could act as a general base that could maintain catalytic activity. Overexpression of the H303I mutant yielded 3.9 mg of enzyme per liter of media, the H303D mutant yielded 3.3 mg of enzyme per liter of media, and the H303E mutant yielded 3.0 mg/L of media. Overexpression of wildtype LOX yielded 4.5 mg/L of media, which is a slight improvement from previous yields. Total copper incorporation for H303I was calculated to be 68% and no copper was detected for the H303D and H303E mutants. As LOX requires the self-processed cofactor lysyl tyrosyl quinone (LTQ) for activity, total LTQ content was obtained by reacting the enzyme with phenylhydrazine and using the previously reported extinction coefficient of 15.4 mM/cm. LTQ content for the wildtype enzyme was determined to be 92%, for H303I the total LTQ content was determined to be 36%, and no LTQ was detected for the H303D and H303E mutants. No catalytic activity was detected for any mutants when compared to the wildtype which has a previously reported activity of 0.11 U/mg. Comparison of excitation–emission matrices (EEM) of each of the mutants as compared to the wildtype indicate that all the mutations cause a change in the internal environment of the enzyme, albeit to varying degrees, as evidenced by the observed shifts.
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Abbreviations
- LOX:
-
Lysyl oxidase
- ECM:
-
Extracellular matrix
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Acknowledgements
The authors wish to thank Dr. Danielle Solano and Luz Perez from the Solano research group at CSU Bakersfield for synthesizing and providing us with the LP-1-2 inhibitor.
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Oldfield, R.N., Johnston, K.A., Limones, J. et al. Identification of Histidine 303 as the Catalytic Base of Lysyl Oxidase via Site-Directed Mutagenesis. Protein J 37, 47–57 (2018). https://doi.org/10.1007/s10930-017-9749-3
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DOI: https://doi.org/10.1007/s10930-017-9749-3