Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

Background: Lysyl oxidases (LOX) were studied mostly in mammals, whereas properties of recently found homologs in prokaryotic genomes remain enigmatic. Methods: LOX gene from Haloterrigena turkmenica has been cloned by PCR in a E. coli expression vector. Protein purification has been done using metal affinity chromatography under denaturing conditions followed by refolding. Catalytic activity has been fluorometrically a release of hydrogen peroxide coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. Results: H. turkmenica LOX (HTU-LOX) may be successfully expressed in E. coli with a high yield. However, full-length protein gives no catalytic activity. On the other hand, a deletion of putative signal peptide allows the protein to be refolded into an active enzyme. Further deletion until the boundary of the catalytic C-terminal domain greatly increases the activity. Refolding is optimal at pH around 6.0, with addition of Cu²⁺, and surprisingly does not respond to changing concentration s of NaCl. The active HTU-LOX is sensitive to the lysyl oxidase inhibitor β-aminopropionitrile. HTU-LOX is active towards usual substrates of mammalian LOX such as lysine-containing basic peptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed specificity of the former. HTU-LOX readily oxidizes various amines including such compounds as taurine and glycine. Moreover, it is active also towards aminoglycoside antibiotics. Benzyl amine is a poor substrate for HTU-LOX. H. turkmenica cells or culture medium do not contain any detectable amine oxidase activity. Polyclonal antibodies against HTU-LOX detect a band among H. turkmenica proteins with the molecular weight corresponding to the unprocessed enzyme. Conclusion: H. turkmenica contains a lysyl oxidase gene that may give active recombinant enzyme with important biochemical features conserved, for example, sensitivity to β-aminopropionitrile. However, its function in the host may be cryptic. Significance: This is the first report on some properties of lysyl oxidase that originated from a horizontal transfer event into Archea.