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Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica.

Abstract：

Background:Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic. Methods:LOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl 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. Results:Cultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with 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 in H. turkmenica cells. Conclusion:H. turkmenica contains 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. Significance:This 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.

journal_name

PeerJ

journal_title

PeerJ

authors

Pestov NB,Kalinovsky DV,Larionova TD,Zakirova AZ,Modyanov NN,Okkelman IA,Korneenko TV

doi

10.7717/peerj.6691

subject

Has Abstract

pub_date

2019-04-05 00:00:00

pages

e6691

issn

2167-8359

pii

6691

journal_volume

pub_type

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Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica.