Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae*

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.

2). These hydroperoxides are precursors of signal molecules in animals, plants, and fungi. They may take part in inflammation, asthma, cancer development, and the chemical warfare between plants, fungi, and other microorganisms (3,4). The LOX mechanism is initiated with hydrogen abstraction from a bis-allylic carbon of the 1Z,4Z-pentadiene of fatty acids. This is followed by oxygen insertion, which usually produces cis-transconjugated hydroperoxy fatty acids (1,2). Plant, mammals, and a few prokaryotes express FeLOX, whereas both MnLOX and FeLOX occur in plant pathogenic fungi (5)(6)(7)(8).
All LOX belong to the same gene family, but plant FeLOX, mammalian FeLOX, and fungal FeLOX and MnLOX form separate subfamilies (5,8). The prototype MnLOX is secreted by the take-all fungus of wheat, Gaeumannomyces graminis (7). The evolution of this enzyme, 13R-MnLOX, and five members of the MnLOX subfamily are illustrated in a phylogenetic tree together with pro-and eukaryotic LOX, including fungal FeLOX (Fig. 1A).
The importance of residues in the active site of LOX has been confirmed by site-directed mutagenesis and recently with three-dimensional structures of bound substrates or inhibitors (13,24). The regio-and stereospecificity of LOX can be a result of different head-to-tail orientations of the substrate, the depth of the active site, residues positioning the hydrogen for abstraction close to the catalytic metal, and oxygen channels (1,2). The MnLOX and FeLOX reaction mechanisms differ in two principal ways as follows: (i) hydrogen abstraction and oxygen insertion occur in a suprafacial manner in at least five MnLOX and antarafacially in all FeLOX (Fig. 1B) (6,21,22,26); (ii) MnLOX are able to oxidize and rearrange bis-allylic hydroperoxides, a reaction that FeLOX only catalyze to a very low rate (27)(28)(29). This difference is possibly related to the redox properties of protein-bound iron and manganese and to structural factors. The adjustment of the different redox potentials iron and manganese is an unresolved issue as well as the metal preference of MnLOX that occurs even though the intracellular iron concentration is higher than the manganese concentration (6,30).
The three-dimensional structure of MnLOX may provide important information on the catalytic mechanism, metal selection, and will allow a comparison between MnLOX and FeLOX. Mo-MnLOX has recently been expressed in Pichia pastoris as a stable enzyme in high yields (22). We therefore selected Mo-MnLOX for three-dimensional structure analysis due to its suitable biochemical properties and biological importance in rice blast disease. We now report the crystallization and 2.0 Å resolution structure of Mo-MnLOX.
Expression and Purification-The Mo-MnLOX precursor consists of 619 amino acids, including a predicted secretion signal of 16 amino acids (GenBank TM accession number ALE27899). Mo-MnLOX without the secretion signal was cloned in the pPICZ␣ expression vector in-frame with the yeast ␣-secretion signal and was expressed in P. pastoris as described (17). Large amounts of enzyme (70 mg/liter) were obtained by expression in a bioreactor for 3-4 days. The secreted enzyme constituted of 603 amino acids with two additional amino acids (Glu and Phe) from the expression vector at the N-terminal end. Mo-MnLOX was purified essentially as described (27). The enzyme (in the expression medium with added 136 g of (NH 4 ) 2 SO 4 per liter and pH adjusted to 6.8 with 10 M KOH) was captured by hydrophobic interaction chromatography (30 ml of butyl-Sepharose CL-4B), washed with 25 mM KHPO 4 (pH 6.8), 1 M (NH 4 ) 2 SO 4 , and eluted with 25 mM KHPO 4 (pH 6.8) using ÄKTA FPLC.

), and
A. fumigatus (EAL84806). The tree was generated by MEGA6 (25) as described (38). B, overview of the oxidation of linoleic acid to hydroperoxides by FeLOX and MnLOX. Both enzymes catalyze the abstraction of the pro-11S hydrogen. The formed radical is delocalized over the pentadiene, and oxygen is typically inserted in an antarafacial way at the 13S or 9R positions by FeLOX, and in a suprafacial way at the 13R, 9S and 11S positions by MnLOX. Note that if the fatty acid enters in the reverse orientation in the catalytic channel, FeLOX can abstract the pro-11R hydrogen and form hydroperoxides with 9S and 13R configuration. sylated with ␣-mannosidase (Sigma) and endoglycosidase H (Sigma) in a protein ratio of 1:40 (w/w) at 21°C overnight. The deglycosylated LOX (in 25 mM HEPES (pH 7.0), 0.1 M NaCl) was purified by gel filtration (Superdex-200 HiLoad 26/600). Fractions with LOX activity were pooled and concentrated to 8 -14 mg/ml by diafiltration (Amicon Ultra 10K) and analyzed by SDS-PAGE.
Site-directed Mutagenesis-Site-directed mutagenesis was performed by whole plasmid PCR technology with Pfu polymerase (16 cycles) according to the QuickChange protocol (Stratagene). 10 ng of the expression vector pPICZ␣A with the open reading frame of Mo-MnLOX served as a template (27). The desired substitutions, R525A and F526L, were introduced with oligonucleotide primers (44 nucleotides). The PCR products were analyzed by agarose gel electrophoresis to confirm amplification of the desired product by digestion of the template DNA with DpnI (37°C, 2 h). All mutations were confirmed by sequencing before expression (Rudbeck Laboratory, Uppsala University). Transformants were obtained after linearization with SacI, transformation of P. pastoris (strain X-33), and selection on yeast peptone dextrose agar plates with phleomycin (100 g/ml) at 28°C (28). Transformed cells were stored as glycerol stocks at Ϫ80°C, and expression was performed in laboratory bench shakers as described (22). The mutated enzymes were captured by hydrophobic interaction chromatography as above, and protein expression was confirmed by SDS-PAGE.
Thermostability-The thermostability of Mo-MnLOX before and after deglycosylation was determined with SYPRO Orange (Invitrogen) and a thermocycler (CFX Connect real time PCR, Bio-Rad). Fluorescence was monitored using the FAM filter (excitation 495 nm; detection 520 nm) as the temperature was gradually increased from 20 to 90°C (1.5°C/min).
Samples were prepared in triplicate and contained 5 M Mo-MnLOX and SYPRO Orange (final dilution 1:700 of 5000 concentrates) in 25 mM HEPES (pH 7.0), 100 mM NaCl, in a total volume of 30 l. Data evaluation and melting temperature determination were performed using the Bio-Rad CFX manager software.
Crystallization-Initial crystallization screens were performed with sitting-drop vapor diffusion in 0.3-l drops in a 96-well plate with aid of a Mosquito crystallization robot (TPP Labtech, Cambridge, UK). Crystal optimization was carried out by hanging drop vapor diffusion by mixing 1 l of 8.5 mg/ml protein with a 1-l reservoir solution (0.1-0.2 M ammonium citrate dibasic (pH 6.5), 10 -16% w/v PEG-3350) in a 15-well plate.
Data Collection and Processing-Data were collected with the focus of achieving a high sulfur and manganese signal at a wavelength of 1.77 Å at 100 K; one dataset was collected at beam line ID29 at the European Synchrotron Radiation Facility, Grenoble, France, and five additional datasets were collected for the same purpose at beam line I02 at the Diamond Light Source, Oxfordshire, UK.
The datasets were processed using XDS (32), and the integrated data were scaled using AIMLESS (33). A set of 5% of the reflections was set aside and used to calculate the quality factor R free . None of the datasets provided sufficient anomalous signal to find the manganese and the sulfur sites. To increase the anomalous signal, all the datasets were analyzed for crystal isomorphism using BLEND (34). Four of the XDS integrated datasets were merged and scaled with R meas and R p.i.m. values of 0.152 and 0.017 and multiplicity of 77.4.
Structure Determination and Refinement-The positions of manganese and sulfur atoms were determined using the HKL2MAP graphical interface with SHELXC, SHELXD, and SHELXE (35,36). SHELXC showed an anomalous signal extending to 3.5 Å resolution. Two manganese and 15 sulfur sites were identified in SHELXD. The correctness of the solution was confirmed by SHELXE. Single anomalous dispersion phasing was performed by phenix.autosol from the Phenix suite (37), using the sites obtained by HKL2MAP and the merged dataset. The phases obtained from SHELX and the protein sequence were submitted to phenix.autosol, and phenix. autobuild (37) was able to build 871 out of 1210 residues of the two molecules in the asymmetric unit, with R work 0.37 and R free 0.39. By using a single dataset to 2.0 Å resolution, we could build 1136 out of 1210 residues with R work 0.17 and R free 0.21. Model evaluation and manual model building were performed in Coot (38). Refinement was performed with phenix.refine (39). Model quality was evaluated with MOLPROBITY (40). 97% of the residues were in favored regions of the Ramachandran plot. Statistics of data collection, processing and model building are presented in Table 1.
Miscellaneous Methods-SDS-PAGE was performed as described (27). Sequences of FeLOX and MnLOX were aligned with the ClustalW program, and a phylogenetic tree was constructed by MEGA6 with bootstrap tests of the resulting nodes (41). All figures were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).

Results
Deglycosylation-Mo-MnLOX retained more than 50% of the enzyme activity after deglycosylation, and the three-dimensional structure discussed below showed that N-acetylglycosamine residues remained at Asn-72, Asn-150, and Asn-535. The deglycosylation process decreases the melting temperature of Mo-MnLOX from 60 to 56°C. The protein unfolding in response to temperature (assayed with SYPRO Orange (22)) indicated that the enzyme solution was not fully homogeneous (supplemental Fig. S1, A and B).  Fig. S1C). Crystals were cryo-protected in reservoir solution with 15% (v/v) glycerol and vitrified in liquid N 2 prior to data collection.
X-ray Diffraction Analysis-The crystals of Mo-MnLOX were relatively insensitive to radiation, and 4800 images were collected with 0.15°oscillation. The best dataset had a completeness of 99.6% and an average multiplicity of 25.3. The crystals of Mo-MnLOX diffracted to 2.0 Å. Mo-MnLOX contains one manganese atom in the active site and 15 sulfur atoms from 3 Cys and 12 Met residues. This made it possible to solve the structure by single anomalous dispersion phasing using both manganese and sulfur atoms as anomalous scatterers. We therefore collected data at 1.77 Å to mitigate the absorption effect at longer wavelengths while still being able to collect a useful sulfur signal. Because the K ␣ absorption edge of a manganese is at ϭ 1.88 Å, the anomalous signal for manganese (fЉ ϭ 3.45 electrons) at ϭ 1.77 Å becomes an additional source of anomalous signal, which facilitated the phase determination. The anomalous signal is weak for sulfur (fЉ ϭ 0.7 electrons) at ϭ 1.77 Å. It was therefore crucial to have high redundancy data to enhance the signal to noise ratio. Four datasets were merged that resulted in a multiplicity of 77.4 and an anomalous signal that enabled us to determine the position of the manganese and sulfur atoms.
The crystals belong to space group P2 1 2 1 2 1 with unit cell dimensions as follows: a ϭ 70.7 Å, b ϭ 111.4 Å, and c ϭ 171.2 Å. The solvent content was 47% with two molecules in the asymmetric unit with the average C ␣ root-mean-square (r.m.s.) deviation of 0.147 Å. The final model was refined to R work of 0.17 and R free of 0.21. We were able to build all the residues except for the 37 N-terminal residues of the expressed protein (EFV . . . PEL), possibly due to its flexibility, as well as N-acetylglucosamine groups at Asn-72, Asn-150, and Asn-535.
Crystal Structure of Mo-MnLOX-Mo-MnLOX lacks the PLAT domain found in many FeLOX and phospholipases (2). An illustration of the overall Mo-MnLOX structure is presented in Fig. 2A. The structure is composed of 21 ␣-helices and 7 small ␤-sheets. The helices ␣9 and ␣10 combined are designated the broken arched helix, and it is sheltering the active site (light green, Fig. 2A). The most striking difference to FeLOX is the orientation of helix ␣2 with 11 turns between residues 79 and 117 (blue helix, Fig. 2B). This long helix is slightly archshaped and runs over the whole length of the protein. Its orientation in animal and plant LOX varies, and in plants it has been reported to be mobile to allow access to the active site, as illustrated by a comparison of Mo-MnLOX with human 5-LOX, sLOX-1, and 15S-LOX of P. aeruginosa (Fig. 2B) (2). This helix is found in all known LOX structures and harbors several invariant hydrophobic residues (2). A structure-based sequence alignment between 8R-LOX (4QWT; the 8R-LOX domain of the allene oxide synthase-LOX fusion protein) and Mo-MnLOX shows conservation of most ␣-helices (supplemental Fig. S2).
A small loop of 5 residues connects helices ␣17 and ␣18 and harbors the manganese ligand, Asn-473 (Fig. 4, A and B). This loop likely brings Asn-473 to a flexible position near the catalytic metal. The side chain oxygen of Gln-281 forms hydrogen bonds to the amino group of Asn-473 with a distance of 2.91 Å (Fig. 4C). This Gln residue is conserved in all FeLOX and MnLOX. There is also a hydrogen bond between the C-terminal carboxyl (Val-605) and the catalytic water (Fig. 4C).
Substrate Channel-There is a solvent-accessible channel leading into the catalytic center of Mo-MnLOX (Fig. 5A). Arg-525 at the entrance is positioned at helix ␣19. This Arg is conserved in five out of the six confirmed MnLOX in Fig. 1A (except in MnLOX of Fusarium oxysporum). Arg-525 is positioned close to Arg-182 of 8R-LOX when the two structures are superimposed. Arg-525 likely forms a salt bridge with the carboxyl end of the substrate fatty acid (Fig. 5B) in analogy with Arg-182 (2). The entrance is also defined by residues from helix ␣2 (Trp-93, Val-98, Ser-101, and Phe-105), helix ␣9 (Leu-326), and helix ␣11 (Val-350).
Leu-331 is situated at the bottom of the arched helix where it shelters the active site, and the corresponding residue of  8R-LOX, Leu-431, has been shown to clamp the substrate in the active site (2,15,24). It appears to play the same role in Mo-MnLOX (Fig. 6). The side chain of the next residue, Phe-332, points into the hydrophobic channel and might shield one side of the pentadiene from oxygenation. This Phe residue is conserved in all MnLOX, but not in FeLOX, which have either Ile or Val at this position (Table 3).
Phe-526 is also conserved in MnLOX, whereas the corresponding residue in FeLOX is a conserved Leu residue ( Table  3). The distance between the side chains of Leu-331 and Phe-526 is only 3.9 Å. The substrate could be clamped by these residues and bent to allow oxygen to access the 11S position after the hydrogen abstraction. The distance between the two corresponding Leu residues in 8R-LOX is similar, but with the substrate in the active site Leu-627 of 8R-LOX is bent backwards, and the distance is increased to 5.2 Å (24).
Oxygen Access to the Catalytic Center-MnLOX utilize suprafacial hydrogen abstraction and oxygenation in contrast to the antarafacial oxidation mechanism of FeLOX (6,21,22,26). This implies that O 2 can access the pentadienyl radical from the same side as the catalytic complex, Mn 3ϩ OH Ϫ (cf. Fig.  1B). A possible oxygen channel has been identified in several three-dimensional structures of FeLOX (2,10,24). The Coffa-Brash determinant, Gly-427 of coral 8R-LOX, appears to be in a critical position in its oxygen channel (2). No equivalent channel could be found in Mo-MnLOX, and the corresponding Gly-327 residue may have little influence on the position of oxygenation (42). There are two pockets in the Mo-MnLOX substrate channel that could harbor oxygen if it enters via the substrate channel (Fig. 6). It is tempting to speculate that these pockets could explain the stereospecific oxygenation of all three positions of the pentadiene radical.
Site-directed Mutagenesis-The structure discussed above suggested that Arg-525 and Phe-526 might be of structural importance for tethering of the carboxyl group and for oxygenation, respectively. We examined the following two mutations: R525A and F526L. Protein expression was confirmed by SDS-PAGE after protein isolation by hydrophobic interaction chromatography.
The mutant R525A transformed 16, 36, and 100 M 18:3n-3 to small amounts of 11-HPOTrE without apparent substrate inhibition (24). 11-HPOTrE was detected by RP-HPLC-MS/MS analysis (supplemental Fig. S3). The marked reduced catalytic activities could be in agreement with the proposed   The Arg-182 of 8R-LOX has been found to tether the carboxylate of the substrate. The Arg-525 of MnLOX is provided by a helix closer to the C terminus, but these two Arg residues seem nevertheless to play similar roles in the tethering of the carboxyl group.
function of Arg-525 in tethering the carboxyl group of 18:2n-6 and 18:3n-3. We also examined 9S-HPOTrE as a substrate of R525A. A substantial fraction of 9S-HPOTrE was transformed to 9,16-DiHOTrE as shown in Fig. 7. RP-HPLC analysis showed that it consisted mainly of the expected 9S,16S diastereoisomer (22). We conclude that Arg-525 is not essential for the lipoxygenation of 9S-HPOTrE.

Discussion
We report as our main finding the first three-dimensional structure of MnLOX. This structure relates to three fundamental differences between MnLOX and FeLOX as follows: (i) the coordinating spheres of Mn 2ϩ and Fe 2ϩ and the metal preferences; (ii) adjustment of the redox potentials of protein-bound Mn 2ϩ /Mn 3ϩ and Fe 2ϩ /Fe 3ϩ and the catalytic base by hydrogen bonds, and (iii) the active sites and the supra-and antarafacial oxygenation mechanisms of MnLOX and FeLOX, respectively. An overview of the active site is shown in Fig. 8, and a comparison of important residues with FeLOX is presented in Table 3. The overall amino acid sequence identity of Mo-MnLOX and 8R-LOX is about 23% with an overall r.m.s. deviation of 3.48 Å.
Metal Coordinating Sphere-Mn 2ϩ is bound in a distorted octahedral configuration in analogy with Fe 2ϩ in eukaryotic and prokaryotic LOX by three His residues, an Asn residue, and the carboxylate of the C-terminal residue ( Fig. 8; Table 3). The metal ligand residues of MnLOX and FeLOX are thus identical except for the replacement of the C-terminal Ile residue with Val in 5 out of 6 MnLOX (Fig. 1A), but the ligands are not identical in space. This is shown by a comparison of the metal ligands of Mo-MnLOX with coral 8R-LOX (Fig. 3A), which align with an r.m.s. deviation of 0.57 Å. The largest differences are found between the Asn residues and the C-terminal Ile or Val residues (Fig. 3A). In contrast, the F-coordinating residues of coral 8R-LOX, sLOX-1, and 15S-LOX of P. aeruginosa can be aligned almost perfectly with an r.m.s. deviation of 0.23-0.29 Å (Fig. 3B) (2).
The Asn-473 ligand of Mo-MnLOX is positioned on a short loop, whereas the corresponding Asn of 8R-LOX and other FeLOX is positioned on an ␣-helix (Fig. 4, A and B). This appears to be one of the most striking differences between the coordination spheres. Oxidation of Mn 2ϩ to Mn 3ϩ may lead to Jahn-Teller distortion from the octahedral coordination of Mn 2ϩ (43). This might be facilitated by the position of Asn-473 on a relatively flexible loop in comparison with position on an ␣-helix.
Fe 2ϩ is usually present in a much larger intracellular concentration than Mn 2ϩ (30). The incorporation of Mn 2ϩ by apoproteins therefore likely occurs in specific cellular compartments, which are enriched in Mn 2ϩ (30). Whether the three-dimensional differences between the coordinating spheres of MnLOX and FeLOX also can affect metal selection will await future studies.
Adjustment of Redox Potentials-FeLOX and MnLOX catalyze the same enzymatic reactions, and their redox properties are therefore likely similar, about 0.6 V (44). As far as is known, manganese-substituted FeLOX are catalytically inactive (9,38). Two differences between MnLOX and FeLOX are the capacity of Mo-MnLOX to catalyze ␤-fragmentation of 11-hydroperoxides of 18:2n-6 and its prolonged catalytic lag phase (22,27,28). Hydrogen bonds to the catalytic center and steric factors likely adjust the redox potential of Mn 2ϩ OH 2 close to that of Fe 2ϩ OH 2 in analogy with manganese and iron superoxide dismutases (45). The catalytic water forms an almost identical hydrogen bond with the carboxylate of the C-terminal Val and Ile residue of Mo-MnLOX and coral 8R-LOX, respectively, but there were no additional hydrogen bonds to the catalytic water. We therefore examined the network of hydrogen bonds to the manganese ligands and to the second coordinating sphere, respectively (Figs. 4C and 8). A hydrogen bond likely occurs between the metal coordinating Asn-473 and Gln-281 (2.8 Å) of Mo-MnLOX. A hydrogen bond was also noted between Ser-604 and Gln-474 (2.8 Å), but site-directed mutagenesis of the corresponding Gln residue of 13R-MnLOX did not abolish the catalytic activity (25). The tuning of the redox potential of protein-bound Mn 2ϩ /Mn 3ϩ will need further investigation. This will include further analysis of the hydrogen bond network.
Active Site and the Oxygenation Mechanism-The deduced substrate channel of Mo-MnLOX appears to be similar to the U-shaped channel of coral 8R-LOX and related FeLOX (2). The substrate channel of Mo-MnLOX is solvent-exposed (Fig. 5A), and its interior has spacious pockets close to the presumed position of the pentadiene for hydrogen abstraction and oxygenation (Fig. 6). Arg-525 likely tethers the carboxylate of the substrate in the same way as Arg-182 of 8R-LOX (Figs. 5 and 8). Replacement of Arg-182 of 8R-LOX with Ala led to a dramatic change in the kinetic properties due to striking substrate inhibition (24). The R525A mutant transformed 18:3n-3 to only small amounts of 11-HPOTrE, but it oxidized 9S-HPOTrE to 9S,16S-DiHOTrE more efficiently (Fig. 7). Arg-525 likely also interacts with the carboxyl group of C 18 and C 20 fatty acids. The oxidation of 20:2n-6, 20:3n-3, and 22:5n-6 suggests sufficient space in the active site to allow productive configurations. In analogy with FeLOX, the depth of the substrate channel is likely controlled by Phe-342 at the position of the Sloane determinant ( Fig. 8; Table 3) and by Phe-353 (not shown in Fig. 8).
Two residues, Phe-332 and Phe-526, may directly influence the stereospecific oxygenation of fatty acids. Phe-332 is positioned in the active site above the catalytic metal and likely holds the substrate in place and might shield the opposite side for oxygen insertion (Fig. 8). Mutagenesis of the corresponding Phe-337 residue in 13R-MnLOX to Ile, which is found at this position of sLOX-1 and other FeLOX (Table 3), switched the oxygen insertion in relation to hydrogen abstraction from suprafacial to mainly antarafacial (46). Phe-526 is also positioned near the catalytic metal and might position the substrate for oxygenation. Site-directed mutagenesis of Phe-526 to Leu resulted in loss of oxidation of 18:3n-3 but retention of the oxidation at C-16 of 9S-HPOTrE. The altered LOX activity suggested that this residue could be essential for catalysis, but further steric analysis of this mutant could not be performed as 18:3n-3 was not oxidized. The three-dimensional structure of Mo-MnLOX with a substrate or a substrate mimic will be needed to exactly define the structural importance of the Phe-526 residue.

Conclusion
We report the three-dimensional crystal structure of MnLOX of the rice blast fungus M. oryzae. The results confirm that the metal ligands of MnLOX and FeLOX are essentially conserved but with geometric differences between the coordinating spheres. Arg-525 likely tethers the carboxyl group of the substrate, and a pair of conserved Phe residues near the catalytic center of MnLOX might be key contributors to the unique suprafacial reaction mechanism.
Author Contributions-A. W. purified and crystallized the protein, determined the x-ray structure, prepared the figures, and wrote the paper. E. H. O. initiated the study, wrote the paper, and prepared the figures. S. K. provided assistance in crystallization, data collection, and interpretation. Y. C. determined the x-ray structure together with A. W., prepared the figures, and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.