Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center

The FEBS Journal
Teerawit WaratrujiwongPornsawan Visoottiviseth

Abstract

Purple acid phosphatases (PAPs) from sweet potato (sp) have been classified on the basis of their primary structure and the dinuclear metal center into isoforms spPAP1 [Fe(III)-Zn(II)] and spPAP2 [Fe(III)-Mn(II)]; for spPAP3 only the cDNA is known. With the aim of unraveling the character of the dinuclear metal center we report here the characterization of this isoform at the protein level. We cloned spPAP3 cDNA in a baculovirus and overexpressed this enzyme in Sf9 insect cells. Preparation of recombinant spPAP3 in two steps afforded pure enzyme with yields of 4.5 mg.L(-1) culture medium. This enzyme is a dimeric, disulfide-linked PAP of 110 kDa, similar to known PAP isoforms from higher plants. Enzymatic studies and spectroscopic properties (max. absorption at 550-565 nm) indicated a diiron enzyme; quantitative and semiquantitative metal analysis using ICP-OES and TOF-SIMS, respectively, revealed the presence of only iron in purified spPAP3. Metal replacement in the second metal-binding site upon preparation of the semiapo-enzyme with Fe(II), Zn(II), or Mn(II) showed highest activities with Fe(II). The data show that recombinant spPAP3 has a diiron metal center. Site-directed mutagenesis was conducted to check catalytic effici...Continue Reading

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Citations

Jan 12, 2011·Plant Physiology·Giuseppe DionisioHenrik Brinch-Pedersen
Sep 3, 2008·Phytochemistry·Rumi KaidaTakako S Kaneko
Jul 15, 2011·Enzyme Research·Claudia Fernanda DickJosé Roberto Meyer-Fernandes
Oct 19, 2006·Analytical Biochemistry·Mariusz Olczak, Teresa Olczak
Mar 21, 2007·Archives of Biochemistry and Biophysics·Mariusz Olczak, Teresa Olczak

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