scholarly journals Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6691
Author(s):  
Nikolay B. Pestov ◽  
Daniel V. Kalinovsky ◽  
Tatyana D. Larionova ◽  
Alia Z. Zakirova ◽  
Nikolai N. Modyanov ◽  
...  
Keyword(s):  
Western Blotting ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Polyclonal Antibodies ◽  
Primary Amines ◽  
Oxidase Gene ◽  
E Coli ◽  
Haloterrigena Turkmenica ◽  
Amine Oxidase Activity

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.

scholarly journals Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

2017 ◽  
Author(s):  
Nikolay B Pestov ◽  
Daniel V Kalinovsky ◽  
Tatyana D Larionova ◽  
Alia Z Zakirova ◽  
Nikolai N Modyanov ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Polyclonal Antibodies ◽  
Oxidase Inhibitor ◽  
High Yield ◽  
Transfer Event ◽  
Oxidase Gene ◽  
E Coli ◽  
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.

scholarly journals Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

2017 ◽  
Author(s):  
Nikolay B Pestov ◽  
Daniel V Kalinovsky ◽  
Tatyana D Larionova ◽  
Alia Z Zakirova ◽  
Nikolai N Modyanov ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Polyclonal Antibodies ◽  
Oxidase Inhibitor ◽  
High Yield ◽  
Transfer Event ◽  
Oxidase Gene ◽  
E Coli ◽  
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.

scholarly journals Extracellular Processing of Lysyl Oxidase-like 2 and Its Effect on Amine Oxidase Activity

Biochemistry ◽  
2018 ◽  
Vol 57 (51) ◽  
pp. 6973-6983 ◽  
Author(s):  
Kazushi Okada ◽  
Hee-Jung Moon ◽  
Joel Finney ◽  
Alex Meier ◽  
Minae Mure
Keyword(s):  
Oxidase Activity ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Amine Oxidase Activity

scholarly journals Lysyl Oxidase Binds Transforming Growth Factor-β and Regulates Its Signaling via Amine Oxidase Activity

2008 ◽  
Vol 283 (49) ◽  
pp. 34229-34240 ◽  
Author(s):  
Phimon Atsawasuwan ◽  
Yoshiyuki Mochida ◽  
Michitsuna Katafuchi ◽  
Masaru Kaku ◽  
Keith S. K. Fong ◽  
...  
Keyword(s):  
Growth Factor ◽  
Oxidase Activity ◽  
Transforming Growth Factor ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Transforming Growth Factor Β ◽  
Amine Oxidase Activity

scholarly journals Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2819-2830 ◽  
Author(s):  
Julie Laliberté ◽  
Simon Labbé
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Nitrogen Source ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Yeast Cells ◽  
Primary Amines ◽  
Sole Nitrogen Source ◽  
Amine Oxidase Activity ◽  
Copper Amine

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao + genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1 + resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2 + failed to produce an active CAO. Using a functional spao1 +–GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Δ null cells exhibited no CAO activity, while ccc2Δ mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Δ and ccs1Δ did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1 + required a functional atx1 + gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Δ cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Δ allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1 + gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

Mitochondrial Oxidation of p-N, N-Dimethylamino-β-Phenethylamine (DAPA)

1975 ◽  
Vol 33 ◽  
pp. 458-459
Author(s):  
W. Allen Shannon ◽  
Hannah L. Wasserkrug ◽  
andArnold M. Seligman
Keyword(s):  
Guinea Pig ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Histochemical Demonstration ◽  
Guinea Pig Heart ◽  
Pig Kidney ◽  
Cytoplasmic Vacuoles ◽  
Liver And Kidney ◽  
Mitochondrial Oxidation ◽  
Amine Oxidase Activity

The synthesis of a new substrate, p-N,N-dimethylamino-β-phenethylamine (DAPA)3 (Fig. 1) (1,2), and the testing of it as a possible substrate for tissue amine oxidase activity have resulted in the ultracytochemical localization of enzyme oxidase activity referred to as DAPA oxidase (DAPAO). DAPA was designed with the goal of providing an amine that would yield on oxidation a stronger reducing aldehyde than does tryptamine in the histochemical demonstration of monoamine oxidase (MAO) with tetrazolium salts.Ultracytochemical preparations of guinea pig heart, liver and kidney and rat heart and liver were studied. Guinea pig kidney, known to exhibit high levels of MAO, appeared the most reactive of the tissues studied. DAPAO reaction product appears primarily in mitochondrial outer compartments and cristae (Figs. 2-4). Reaction product is also localized in endoplasmic reticulum, cytoplasmic vacuoles and nuclear envelopes (Figs. 2 and 3) and in the sarcoplasmic reticulum of heart.

Acrolein produced from polyamines as one of the uraemic toxins

2003 ◽  
Vol 31 (2) ◽  
pp. 371-374 ◽  
Author(s):  
K. Sakata ◽  
K. Kashiwagi ◽  
S. Sharmin ◽  
S. Ueda ◽  
K. Igarashi
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Oxidase Activity ◽  
Culture Medium ◽  
Cellular Proliferation ◽  
Amine Oxidase ◽  
Early Stage ◽  
Chronic Glomerulonephritis ◽  
Toxic Compound ◽  
Amine Oxidase Activity

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.

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