Eur. J. Biochem. zyxwvutsrqpo 247, 826-832 (1997) zyxwvuts 0 FEBS 1997 zyxwvutsrqp Cloning and biochemical characterization of an anionic peroxidase from zyxwvut Zea mays Thomas TEICHMANN, Changhui GUAN, Peter KRISTOFFERSEN, Gerhard MUSTER, Olaf TIETZ and Klaus PALME Max-Delbriick-Laboratoriutn in der Max-Planck-Gesellschaft, Koln, Germany (Received 20 January/27 May 1997) zyxwvutsr - EJB 97 0071/1 We have isolated, cloned and characterized a cDNA from Zea mays L., denoted ZmAPI, coding for an anionic peroxidase. The open reading frame of ZmAP1 starting 72 residues from the 5’ end of the cDNA predicts a 37778 dalton protein of 356 amino acid residues. The protein has high similarity to other peroxidases and contains two peroxidase motifs that carry two highly conserved histidines in the active center. We expressed recombinant ZmAPl protein in E. coli as a fusion with maltose-binding protein. The fusion protein was biochemically active after addition of hemin to the apoprotein. The maize peroxidase ZmAPl has a pH optimum at pH 4.0 and a K,, of 0.2 mM for the substrate 2,2’-azino-bis- zy (3-ethyl-benzothiazolin-6-sulfonic acid) at this pH. In maize seedlings the ZmAPl gene is expressed predominantly in roots, the mesocotyl, the coleoptile and to a lower extent in the node, whereas no expression in the primary leaf was found. In situ hybridization shows that the expression of ZinAPl in the young maize root is confined to the epidermis, hypodermis and the pericycle. Keywords: maize; peroxidase ; expression in Escherichia coli ; elongation ; gene expression. Peroxidases are donor :hydrogen peroxide oxidoreductases of apparent M, of 32000 to 45000. They form a broad class of heme-containing enzymes that act on a wide variety of sub- strates. They receive two oxidation equivalents from hydroper- oxide with which a broad range of aromatic hydrogen donors is oxidized and subsequently released as oxidized radicals. Accord- ing to this reaction mechanism they are involved in a wide range of functions such as initiation of polymerization and depolymer- ization reactions, removal of toxic hydroperoxides and in electron transport or in diverse biosynthetic and degradative pathways using hydrogen peroxide as the electron acceptor. Peroxidases have been found in microorganisms, yeast, ani- mals and plants. Plant peroxidases have been shown to play a role in lignin biosynthesis (Harkin and Obst, 1973), extensin polymerization, defense reactions against pathogen attack (La- grimini et al., 1987) response to wounding (Sherf and Kolattu- kudy, 1993), control of stem elongation (Jupe and Scott, 1992), control of development and differentiation (van Engelen and deVries, 1992), degradation of the plant hormone auxin (Hoyle, 1977) and detoxification of SO, (Kubo et al., 1995). It is thought that different isoenzymes mediate these diverse roles in the rele- vant plant cells, tissues and organs and that some of these en- zymes will be of general importance for biotechnological appli- cations. Several different peroxidase isoenzymes have been found and classified as anionic, neutral and cationic enzymes accord- Correspondence to T. Teichmann, Max-Delbriick-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-LinnC-Weg 10, D-50829 Koln, Germany Fax: +49 221 506 2613. E-mail: teichman @ mpiz-koeln.mpg.de Abbreviation. ER, endoplasmic reticulum. Enzyme. Peroxidase (EC 2.11.1.7.) Note. The nucleotide sequence data published here have been sub- mitted to the EMBL sequence data bank and are available under acces- sion number Y 13905. ing to their isoelectric point. Anionic peroxidases have been shown to mediate the cross-linking of cell wall proteins, to be involved in making cell walls effective barriers to diffusion by suberization and to play an important role in lignification. As a first step towards the characterization of anionic peroxidases from maize we have isolated and cloned a cDNA encoding an anionic peroxidase from Zea mays and studied its biochemical properties after heterologous expression in Escherichia coli. MATERIALS AND METHODS Materials. Zea mays L.; var. Mutin (D/FR 7205034) was obtained from Kleinwanzlebener Saatzucht, Einbeck, Germany. Horseradish peroxidase and 2,2’-azino-bis-(3-ethyl-benzothia- zolin-6-sulfonic acid) was purchased from Boehringer. 3- Amino-9-ethyl-carbazole and bovine hemin was from Sigma. pMal-C2 expression vector and amylose were from New Eng- land Biolabs. [a-32P]dCTP was from Amersham. Factor Xa was purchased from Denzyme Aps. Expression library construction and screening of COS cells. A cDNA library was constructed in the mammalian ex- pression vector pCDM8 and transfected into COS cells as de- scribed previously (Kristoffersen et al., 1996). COS cells ex- pressing peroxidases were identified by staining for peroxidase activity using 3-amino-9-ethyl-carbazole as a substrate. Clone analysis. DNA sequencing. cDNA inserts were iso- lated from pCDM8 after XbaI digestion and subcloned into pBluescript vectors for DNA sequencing. Both strands were se- quenced using an Automated Laser Fluorescent DNA sequencer (ALF, Pharmacia). Northern analysis. Total RNAs from different parts of four- day-old maize seedlings were isolated according to Chomczyn- ski and Sacchi (1987). Poly(A)-rich RNA was extracted using Oligotex from Quiagen. For the northern blot analysis 2 pg of poly(A)-rich RNA was transferred to Hybond N membrane (Amersham) and analyzed with a ”P-labeled DNA probe.