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BackgroundLysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.MethodsLOX open reading frame was cloned fromHaloterrigena turkmenicain anE. coliexpression vector. RecombinantHaloterrigena turkmenicalysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release 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.ResultsCulturedH. turkmenicahas no detectable amine oxidase activity. HTU-LOX may be expressed inE. coliwith a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein inH. turkmenicacells.ConclusionH. turkmenicacontains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.SignificanceThis is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

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.