Lys296 and Arg299 residues in the C-terminus of MD-ACO1 are essential for a 1-aminocyclopropane-1-carboxylate oxidase enzyme activity Ahrim Yoo a,1 , Young Sam Seo b,1 , Jin-Won Jung c , Soon-Kee Sung d , Woo Taek Kim b, * , Weontae Lee c,e, * , Dae Ryook Yang a, * a Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Republic of Korea b Department of Biology, College of Science, Yonsei University, Seoul 120-749, Republic of Korea c Department of Biochemistry and National Research Laboratory of HTSD-NMR and Application, Yonsei University, Seoul 120-749, Republic of Korea d Plant Biotechnology Team, Dongbu Advanced Research Institute, Daejeon 305-708, Republic of Korea e Protein Network Research Center, College of Science, Yonsei University, Seoul 120-749, Republic of Korea Received 6 March 2006; received in revised form 22 August 2006; accepted 24 August 2006 Available online 7 September 2006 Abstract The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe 2+ /H 2 O derived from Petunia hybrida has recently been established by X-ray crystal- lography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30– 15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclu- sively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the posi- tive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Comparative modeling; Molecular dynamics; Site-directed mutagenesis; 1-Aminocyclopropane-1-carboxylate oxidase; C-terminus; Substrate binding 1. Introduction The gaseous plant hormone, ethylene, regulates many processes of plant growth, development, and senescence (Abeles et al., 1992; Yang and Hoffman, 1984). 1-Aminocy- clopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants (Kim and Yang, 1994; Yang and Hoffman, 1984). The first identification of a cDNA clone, pTOM13, encod- ing ACC oxidase was achieved in tomato fruit through an RNA antisense strategy (Hamilton et al., 1990). The deduced amino-acid sequence of pTOM13 shows homolo- gy to that of flavone 3-hydroxylase, which requires both Fe 2+ and ascorbate for its enzyme activity (Hamilton et al., 1991). Thereafter, it was demonstrated that purified ACC oxidase (MD-ACO1) from ripening apples (Malus domestica Borkh.) requires CO 2 as an activator of enzyme activity (Dong et al., 1992). The ACC oxidase is a member of the non-heme iron enzyme family in which most of their members require Fe 2+ and 2-oxoglutarate as a co-substrate. 1047-8477/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2006.08.012 * Corresponding authors. Fax: +82 2 362 9897 (W. Lee). E-mail addresses: wtkim@yonsei.ac.kr (W.T. Kim), wlee@spin. yonsei.ac.kr (W. Lee), dryang@korea.ac.kr (D.R. Yang). 1 These authors contributed equally. www.elsevier.com/locate/yjsbi Journal of Structural Biology 156 (2006) 407–420 Journal of Structural Biology