tailieunhanh - Báo cáo khoa học: Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center

Purple acid phosphatases (PAPs) from sweet potato (sp) have been classi-fied 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 charac-ter of the dinuclear metal center we report here the characterization of this isoform at the protein level. | ềFEBS Journal Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center Teerawit Waratrujiwong1 2 Bernt Krebs3 Friedrich Spener2 and Pornsawan Visoottiviseth1 1 Department of Biology Faculty of Science MahidolUniversity Bangkok Thailand 2 Department of Biochemistry University of Munster Germany 3 Department of Analyticaland Inorganic Chemistry University of Munster Germany Keywords baculovirus purple acid phosphatase semiapo-enzyme site-directed mutagenesis sweet potato Correspondence P. Visoottiviseth Department of Biology Faculty of Science MahidolUniversity Rama VI Road Phyathai Bangkok 10400 Thailand Fax 66 2247 0079 Tel 66 2201 5272 E-mail scpvi@ Website http scbi Received 30 November 2005 revised 9 February 2006 accepted 15 February 2006 doi 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 spPAPl 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 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 .