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The Crystal Structure of Chloroperoxidase: a Heme Peroxidase-Cytochrome P450 Functional Hybrid Munirathinam Sundaramoorthy', James Terner 2 and Thomas L Poulosl*

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The Crystal Structure of Chloroperoxidase: a Heme Peroxidase-Cytochrome P450 Functional Hybrid Munirathinam Sundaramoorthy', James Terner 2 and Thomas L Poulosl* View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector The crystal structure of chloroperoxidase: a heme peroxidase-cytochrome P450 functional hybrid Munirathinam Sundaramoorthy', James Terner 2 and Thomas L Poulosl* 1 Departments of Molecular Biology and Biochemistry and, Physiology and Biophysics, University of California, Irvine, CA 92717, USA and 2Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284-2006, USA Background: Chloroperoxidase (CPO) is a versatile heme- be the binding site for substrates that undergo P450-like containing enzyme that exhibits peroxidase, catalase and reactions. The crystal structure also shows extensive glyco- cytochrome P450-like activities in addition to catalyzing sylation with both N- and O-linked glycosyl chains. halogenation reactions. The structure determination of CPO Conclusions: The proximal side of the heme in CPO was undertaken to help elucidate those structural features resembles cytochrome P450 because a cysteine residue that enable the enzyme to exhibit these multiple activities. serves as an axial heme ligand, whereas the distal side of the Results: Despite functional similarities with other heme heme is 'peroxidase-like' in that polar residues form the enzymes, CPO folds into a novel tertiary structure domi- peroxide-binding site. Access to the heme pocket nated by eight helical segments. The catalytic base, is restricted to the distal face such that small organic required to cleave the peroxide 0-0 bond, is glutamic substrates can interact with an iron-linked oxygen atom acid rather than histidine as in other peroxidases. CPO which accounts for the P450-like reactions catalyzed by contains a hydrophobic patch above the heme that could chloroperoxidase. Structure 15 December 1995, 3:1367-1377 Key words: carbohydrate binding, halogenation, heme enzyme, peroxidase, thiolate ligand Introduction Another important property of both peroxidases and Halogenation of aliphatic molecules gives rise to a wide P450s is accessibility to the heme group. In peroxidases, variety of natural products, some of which, like chloram- substrates are limited to electron-transfer reactions at the phenicol, are of medicinal value [1]. Enzyme-catalyzed heme edge with restricted or no direct access to the halogenation reactions most often involve oxidation of Fe4+=O center in compound I [9]. In sharp contrast, the the halide ion and formation of a C-X bond. The heme P450 heme edge is not accessible and substrates must enzyme, chloroperoxidase (CPO), from the fungus Cal- bind directly adjacent to the Fe4 +=O center for stereo- dariomyces fiimago, participates in the production of the specific hydroxylation [10]. CPO can do both, but natural product caldariomycin (1,1 -dichloro-2,5-dihy- whether this is due to multiple access routes to the heme droxy cyclopentane) [2]. The overall catalytic cycle of remains unknown. The structure of this rather unusual CPO is characteristic of many heme peroxidases (Fig. la). and functionally diverse heme enzyme should help us Although the primary biological function of CPO is 4 chlorination [2], CPO also catalyzes other oxidative reac- (a) Fe3+P+H 2 02 - Fe +=O P +H20 tions characteristic of other heme peroxidases, catalase Compound I and cytochrome P450 (Fig. lb) [3]. Owing to this diver- Fe4+=O P'+ substrate -- Fe4+=O P + substrate' sity, CPO is the most versatile of the known heme Compound II enzymes and has been the subject of extensive investiga- Fe4+=O P + substrate - Fe3+ P +substrate' tions since the enzyme was first described by Morris and + Hager in 1966 [2]. One of the main conclusions from (b) Halogenation RH+ H202 +CI-+ H - R-CI +2H 2 0 these studies is that CPO appears to share properties with Dehydrogenation 2RH + H202 -- R-R + 2H 20 both P450s and heme peroxidases. CPO and P450s share (heme peroxidases) various spectroscopic properties [4,5] which indicate H202 decomposition 2H 202 -02+2H 20 that, like P450, one of the axial heme ligands in CPO (catalase) derives from the sulfur atom of a cysteine residue. In Oxygen insertion R+ H2 02 - R-O+ H2 0 (P450) contrast, heme peroxidases use histidine as a ligand. However, the distal heme pocket in CPO, which forms the peroxide-binding site, is predicted to be formed Fig. 1. (a) Overall catalytic cycle of heme peroxidases. In the first primarily by polar amino acids [6-8]. This is in sharp step, hydrogen peroxide removes one electron from the iron contrast with P450s in which the distal oxygen-binding atom and one from the porphyrin (P) to give the oxyferryl (Fe4+=O) center and a porphyrin rr cation radical [33]. In the sec- site is lined primarily with non-polar groups. Hence, ond step, compound I is reduced by a substrate molecule, and in CPO is thought to have a P450-like proximal pocket and the third and last step of the cycle, a second molecule of sub- a peroxidase-like distal pocket. strate is oxidized. (b) Reactions catalyzed by chloroperoxidase. *Corresponding author. © Current Biology Ltd ISSN 0969-2126 1367 1368 Structure 1995, Vol 3 No 12 to further understand the differences and similarities original DNA sequence indicates that the complete CPO between peroxidases and P450s as well as to probe the gene encodes a protein of 373 amino acid residues [12]. structural basis for the wide range of activities exhibited This 373-residue polypeptide chain is processed to a by CPO. We have crystallized and solved the structure of mature, secreted CPO enzyme containing 299 amino CPO isozyme A in two space groups. The crystal struc- acid residues by two proteolytic cleavages: one removes a ture shows many interesting features, some previously 20 amino acid signal peptide from the N terminus and predicted and some hitherto unknown. the other removes a 52 amino acid peptide from the C terminus. CPO undergoes at least two other post- translational modifications. One of these is glycosylation Results and discussion (discussed later) and the second is cyclization of the Overall structure N-terminal glutamic acid [13]. CPO is synthesized as a The structure has been refined in two space groups, proenzyme with the proteolytic clip between Gln(-1) P21 2121 and C2221, at 1.9 A and 2.1 A respectively and Glul. However, the crystal structure shows that (Table 1). The root mean square deviation (rmsd) residue -1 is still present as the cyclized pyroglutamic acid between the two models is 0.32 A for backbone atoms (designated residue number 0 to allow the previous and 0.4 A for all protein atoms. The deviation of Co numbering scheme to be retained). atoms exceeds 1 A for only four residues at the N termi- nus and two other residues on a surface loop. More than The CPO structure was compared with 420 protein 90% of (4,) angles fall within the most favorable regions structures in the Protein Data Bank using a three-dimen- of Ramachandran plots [11]. Unless otherwise stated, the sional alignment algorithm (DALI [14]). A 30% sequence model described in this paper is the one that was refined identity cutoff was applied. The comparison reveals that in space group P212121 because this crystal form the CPO polypeptide folds into a novel tertiary structure diffracted to higher resolution and had better defined (Fig. 2). The helical content (-50%) is similar to that electron density for the carbohydrate structure. found in other peroxidases and P450s, but the overall topology lacks any resemblance to these other heme Table 1. Model refinement statistics. enzymes. Nevertheless, there are important structural similarities near the active site. As with other heme per- P2 12121 C222 1 oxidases and other proteins, CPO folds into N-terminal and C-terminal domains with the heme sandwiched No. of non-H atoms between the two domains. The fold comprises eight Protein 2316 2316 3 Heme 43 43 ot-helical segments (A-H), three short 10 helices (C', D' Carbohydrates 238 156 and G') and a short antiparallel pair (Table 2). Like Cation 1 1 other heme enzymes, CPO has a proximal helix (helix A) Solvent 190 195 on the side of the heme containing the protein axial Highest resolution 1.9 A 2.1 A R-factor heme ligand, and a distal helix (helix F) that provides cat- Resolution range 8.0-1.9 A 8.0-2.1 A alytic groups required for peroxide activation. Whereas in 2a cutoff 25 373 (18.2%) 23 137 (18.6%) CPO the proximal helix A is approximately perpendicu- No a cutoff 27521 (19.2%) 25 061 (19.6%) lar to the heme plane, in both the peroxidases and P450s Resolution range 30.0-1.9 A 30.0-2.1 A it is parallel to the heme plane. As anticipated from 2a cutoff 25852 (20.9%) 23681 (22.2%) No cr cutoff 28004 (21.9%) 25 607 (23.1%) chemical modification studies [15], Cys29 provides the Free R-factor 22.4% 22.8% thiolate heme ligand. The axial ligand residue is in the Rmsd in bond lengths 0.009 A 0.008 A N-terminal domain in CPO, whereas in other heme ° ° Rmsd in bond angles 1.557 1.488 enzymes it resides in the C-terminal domain. The only Mean coordinate error other two cysteines, Cys79 and Cys87, form a disulfide Luzzati plot [511 0.20 A 0.20-0.25 A SIGMAA [49] 0.22 A 0.26 A bond (Fig. 2). Three prolines (Pro9, Pro230 and Pro292) adopt a cis conformation and one of these (Pro9) is in a classical cis-proline turn [16]. Relative to the cDNA sequence [12], the crystal struc- ture is missing 52 residues at the C terminus. The possi- Proximal heme ligand bility that the C terminus is disordered can be ruled out The polypeptide from residues 26-37 encompasses the because the electron density is well defined in both space cysteine ligand (Cys29) and provides a rigid scaffolding groups and the models exhibit very low temperature for the iron-sulfur interaction.
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