title: The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase.
abstract: Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Bollinger
authors.forename: Doreen E
authors.initials: DE
authors.lastname: Brown
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004331:Drosophila melanogaster / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis* / Q000378:metabolism*; D011809:Quinones; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D011809:Quinones; C100796:lysine tyrosylquinone; D008249:Protein-Lysine 6-Oxidase; D008239:Lysine