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Volume 152, Issue 9

Mechanisms of copper loading on the copper amine oxidase 1 expressed in Free

Abstract

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although 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 genes from were expressed in . Expression of resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of failed to produce an active CAO. Using a functional – fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the , and genes were defective in amine oxidase activity. Likewise, Δ null cells exhibited no CAO activity, while Δ mutant cells exhibited decreased levels of amine oxidase activity, and mutations in Δ and Δ did not cause any defects in this activity. Copper-deprived cells expressing required a functional gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the Δ cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted Δ allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous gene in is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active SPAO1 protein in depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

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Keyword(s): BCS, bathocuproine disulfonic acid , CAO, copper amine oxidase , GFP, green fluorescent protein , HPAO, Hansenula polymorpha amine oxidase , PGK, phosphoglycerol kinase , PTS1, peroxisome targeting signal type 1 , PTS2, peroxisome targeting signal type 2 , TPQ, 2,4,5-trihydroxyphenylalanine quinine and TTM, ammonium tetrathiomolybdate
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2006-09-01
2024-04-19
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Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae
Microbiology 152, 2819 (2006); https://doi.org/10.1099/mic.0.28998-0
/content/journal/micro/10.1099/mic.0.28998-0
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