On the Relationship of Coral Allene Oxide Synthase to Catalase A SINGLE ACTIVE SITE MUTATION THAT INDUCES CATALASE ACTIVITY IN CORAL ALLENE OXIDE SYNTHASE * Received for publication, January 4, 2006, and in revised form, February 28, 2006 Published, JBC Papers in Press, March 2, 2006, DOI 10.1074/jbc.M600061200 Takehiko Tosha ‡1 , Takeshi Uchida ‡2 , Alan R. Brash § , and Teizo Kitagawa ‡3 From the ‡ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan and the § Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602 A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. Although cAOS has a similar heme active site to that of catalase, cAOS is completely lacking in catalase activity. A close look at the hydrogen-bonding possibilities around the distal His in cAOS sug- gested that the imidazole ring is rotated by 180° relative to that of catalase because of the hydrogen bond between Thr-66 and the dis- tal His-67. This could contribute to the functional differences between cAOS and catalase, and to examine this possibility, we mutated Thr-66 in cAOS to Val, the corresponding residue in cata- lase. In contrast to the complete absence of catalase activity in wild type (WT) cAOS, T66V had a modest catalase activity. On the other hand, the mutation suppressed the native enzymatic activity of the formation of allene oxide to 14% of that of WT cAOS. In the reso- nance Raman spectrum, whereas WT cAOS has only a 6-coordi- nate/high spin heme, T66V has a 5-coordinate/high spin heme as a minor species. Because catalase adopts a 5-coordinate/high spin structure, probably the 5-coordinate/high spin portion of T66V showed the catalase activity. Furthermore, in accord with the fact that the CN affinity of catalase is higher than that of WT cAOS, the CN affinity of T66V was 8-fold higher than that of WT cAOS, indi- cating that the mutation could mimic the heme active site in cata- lase. We, therefore, propose that the hydrogen bond between Thr-66 and distal His-67 could modulate the orientation of distal His, thereby regulating the enzymatic activity in cAOS. Allene oxides formed by the enzymatic dehydration of fatty acid hydroperoxides, the lipoxygenase (LOX) 4 products of polyunsaturated fatty acids, are involved in biosynthetic pathways of plants and inverte- brates. The main plant pathway leads to jasmonic acid, which seems to be a physiological signaling molecule for several wound- and pathogen- induced responses (1). The conversion from fatty acid hydroperoxide to allene oxide in plants is catalyzed by a heme containing enzyme called allene oxide synthase (AOS) (2, 3). Plant AOS belongs to a subfamily of the fatty acid hydroperoxide metabolizing cytochrome P450s (P450s) designated as CYP74A (4 – 6). Despite significant sequence homology of plant AOS to those of other P450s, the reaction of plant AOS is different from those of typical P450s (7, 8). Plant AOS does not require the reduc- tants and oxygen, and carries out the homolytic cleavage of the O-O bond in hydroperoxide, although typical P450s utilize two electrons and molecular oxygen for the hydroxylation of its substrate. Koljak et al. (1997) discovered and isolated a cDNA encoding a fusion protein of 8R-LOX and AOS from the Caribbean sea soft coral, Plexau- ra homomalla (9), the first example of an AOS found in animals. The fusion protein consists of a C-terminal 8R-LOX domain (79 kDa) and N-terminal heme-containing AOS domain (43 kDa). As shown in Scheme 1, reaction (i), the 8R-LOX domain initially catalyzes the oxy- genation of arachidonic acid at the 8R position, yielding the 8R-hy- droperoxide of arachidonic acid (8R-HpETE). Then, the AOS domain produces allene oxide from 8R-HpETE (Scheme 1, reaction (ii)). The truncated construct of the AOS domain (cAOS) can catalyze an identi- cal reaction to the AOS domain in the native coral fusion protein (7). Despite the functional similarity to plant AOS, cAOS does not exhibit sequence homology to plant AOS, but shows significant homology to catalase (11% of sequence identity) (9). However, cAOS cannot cata- lyze the dismutation of H 2 O 2 to water and oxygen molecule, namely the catalase reaction (Scheme 2) (7, 9), whereas cAOS, like bovine liver catalase (BLC), reacts with peracetic acid to form a ferryl oxo species with a tyrosine radical followed by the formation of a ferryl oxo porphy- rin -cation radical (compound I) species (10, 11). To clarify the structure-function relationship, especially the reason for the lack of the catalase activity, several spectroscopic techniques including UV/vis, EPR, MCD, and x-ray crystallography were applied to the structural characterization of cAOS (11, 12). As expected from the sequence homology, the spectroscopic data indicated that the heme active site structure in cAOS is quite similar to that of catalase (11, 12). For example, Tyr is a heme ligand of cAOS as observed in catalase (11, 12). On the other hand, as compared with catalase, the recent crystal structure of cAOS showed the lack of hydrogen-bonding network involving the proximal tyrosinate ligand and also the remarkable pla- narity of the heme (12). Oldham et al. (12) proposed that such structural properties would inhibit the reaction with H 2 O 2 , thereby showing no catalase activity in cAOS. In addition, the crystal structures also showed remarkable differences in the heme distal sites between cAOS and catalase as displayed in Fig. 1. Although residues responsible for the catalase reaction such as the distal His and Asn are conserved in cAOS, the hydrogen-bonding pattern of the distal His in cAOS is different from that of catalase (12). In catalase, * This work was supported by a Grant-in-aid for Specifically Promoted Research (14001004, to T. K.) from the Ministry of Education, Culture, Sports, Science and Tech- nology, Japan, and by National Institutes of Health Grant GM-53638 and Pilot Project Grant P30 ES000267 (to A. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a research fellowship from the Japan Society for the Promotion of Sci- ence for young scientists. 2 Present address: Division of Chemistry, Graduate School of Science, Hokkaido Univer- sity, Sapporo 060-0810, Japan. 3 To whom correspondence should be addressed. Tel.: 81-564-59-5225; Fax: 81-564-59- 5229; E-mail: teizo@ims.ac.jp. 4 The abbreviations used are: LOX, lipoxygenase; AOS, allene oxide synthase; cAOS, AOS domain of coral AOS; 8R-HpETE, 8R-hydroperoxy-5Z,9E, 11Z, 14Z-eicosatetraenoic acid; RR, resonance Raman; BLC, bovine liver catalase; HPII, E. coli hydroperoxidase II; WT, wild type. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 18, pp. 12610 –12617, May 5, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 12610 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 18 • MAY 5, 2006 by guest on June 1, 2020 http://www.jbc.org/ Downloaded from