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 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 efficiency at the atomic level. Tyr291 at the substrate-binding site in spPAP3 was mutated to His and Ala, the respective residues found in spPAP1 and spPAP2. Kinetic analysis showed that conversion of Tyr291 to His further optimized the performance of this protein as a diiron enzyme, whereas the Ala mutation weakened the catalytic efficiency regardless of the metal present in the second binding site.
Waratrujiwong
Teerawit
T
Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Krebs
Bernt
B
Spener
Friedrich
F
Visoottiviseth
Pornsawan
P
D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D027723:Ipomoea batatas / Q000201:enzymology*; D007501:Iron / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics
D006023:Glycoproteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase