Properties of a cryptic lysyl oxidase from haloarchaeonHaloterrigena turkmenica
| Autor: | Alia Z. Zakirova, Irina A. Okkelman, Tatyana D. Larionova, Daniel V. Kalinovsky, Nikolay B. Pestov, Nikolai N. Modyanov, Tatyana V. Korneenko |
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| Rok vydání: | 2019 |
| Předmět: |
Signal peptide
Amine oxidase Halophiles lcsh:Medicine Lysyl oxidase Horseradish peroxidase General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 0302 clinical medicine Archeae 030304 developmental biology chemistry.chemical_classification 0303 health sciences integumentary system biology Chemistry General Neuroscience lcsh:R food and beverages Haloterrigena turkmenica Horizontal gene transfer General Medicine enzymes and coenzymes (carbohydrates) Enzyme Biochemistry Polyclonal antibodies Glycine biology.protein General Agricultural and Biological Sciences 030217 neurology & neurosurgery |
| Zdroj: | PeerJ, Vol 7, p e6691 (2019) |
| ISSN: | 2167-8359 |
| DOI: | 10.7717/peerj.6691 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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