Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein

Abstract

The crystal structure of 13R-manganese lipoxygenase (MnLOX) of Gaeumannomyces graminis (Gg) in complex with zonadhesin of Pichia pastoris was solved by molecular replacement. Zonadhesin contains β-strands in two subdomains. A comparison of Gg-MnLOX with the 9S-MnLOX of Magnaporthe oryzae (Mo) shows that the protein fold and the geometry of the metal ligands are conserved. The U-shaped active sites differ mainly due to hydrophobic residues of the substrate channel. The volumes and two hydrophobic side pockets near the catalytic base may sanction oxygenation at C-13 and C-9, respectively. Gly-332 of Gg-MnLOX is positioned in the substrate channel between the entrance and the metal center. Replacements with larger residues could restrict oxygen and substrate to reach the active site. C₁₈ fatty acids are likely positioned with C-11 between Mn²⁺OH₂ and Leu-336 for hydrogen abstraction and with one side of the 12Z double bond shielded by Phe-337 to prevent antarafacial oxygenation at C-13 and C-11. Phe-347 is positioned at the end of the substrate channel and replacement with smaller residues can position C₁₈ fatty acids for oxygenation at C-9. Gg-MnLOX does not catalyze the sequential lipoxygenation of n-3 fatty acids in contrast to Mo-MnLOX, which illustrates the different configurations of their substrate channels.

Lipoxygenases (LOXs) are iron- or manganese-containing dioxygenases (1). These enzymes occur in animals, plants, fungi, and in some prokaryotes (1–3). LOXs oxygenate arachidonic acid (20:4n-6) in man and linoleic and linolenic acids (18:2n-6 and 18:3n-3) in lower organisms to hydroperoxides with stereo and position specificity (1, 3). The hydroperoxides formed from 20:4n-6 can be transformed to leukotrienes and an array of other biological mediators, designated eicosanoids (4). Human LOXs and their products take part in allergic inflammation, inflammation resolution, cancer development, atherosclerosis, and in creating the water barrier of the skin (1, 4, 5). The hydroperoxides formed from 18:2n-6 and 18:3n-3 by plant and fungal LOXs can be further transformed to jasmonates, important regulators of growth, and to other metabolites, designated oxylipins (3). These oxylipins are formed during the pathogenic process and as defense molecules in the chemical warfare between plants, fungi, and bacteria (3).

All LOXs belong to the same gene family with essentially conserved fold and metal ligands. The three dimensional structures of LOXs are available from man and animals including corals, two prokaryotes, and from the rice blast fungus, Magnaporthe oryzae, with catalytic manganese (1, 6, 7). Three His residues, the carboxyl oxygen of the C-terminal Ile or Val residue, and a fifth residue, usually Asn, ligate the metals, which bind water to form a catalytic base (Fe²⁺OH₂ and Mn²⁺OH₂) for hydrogen abstraction (1, 8).

Manganese lipoxygenases (MnLOXs) only occur in ascomycete fungi and form a distinct subfamily (2). These enzymes have been characterized from five pathogens in addition to M. oryzae (9–12). They are named by their catalytic properties with 18:2n-6 as a substrate or by their origin: Gaeumannomyces graminis (13R- or Gg-MnLOX), Magnaporthe salvinii (9S- or Ms-MnLOX), Aspergillus fumigatus (13- or Af-MnLOX), Fusarium oxysporum (11R/13S- or Fo-MnLOX), and Colletotrichum gloesporioides (9S/11S- or Cg-MnLOX) (2, 9–12). Based on sequence homology to Gg-MnLOX, a number of LOX sequences have been denoted as putative MnLOXs at NCBI. We will denote the enzymes by their origin in this report. A phylogenetic tree of the six known and four tentative MnLOXs is shown in Fig. 1A. The amino acids at their C-terminal end are partly conserved and this motif appears to be characteristic of MnLOXs (Fig. 1B), whereas the C terminal of plant and mammalian LOXs ends by the hexamers ProAsnSerIleSerIle and GluAsnSerValAlaIle, respectively.

Fig. 1.

Phylogenetic tree of MnLOXs of ten ascomycetes and overview of amino acids at the C-terminal end. A: Phylogenetic tree. The tree was constructed by MEGA6 (30). The GenBank ID numbers are from top to bottom: G. graminis (AAK81882), M. salvinii (synonym Nakataea oryzae; CAD61974), M. oryzae (ALE27899), C. gloeosporioides (EQB45907), F. oxysporum (EGU80482), Rosellinia necatrix (GAP93054), Botrytis cinerea (CCD55783), A. fumigatus (EDP47436), Penicillium roqueforti (CDM26633), and Diaporthe ampelina (KKY33814). The numbers in bold show the percent sequence identity to Gg-MnLOX. B: The hexamer sequences at the C-terminal end of the ten MnLOXs in (A) are shown as logos, which represent the frequency of a particular amino acid at this position. The sequence logos were created using WebLogo3.0 (http://weblogo.berkeley.edu/logo.cgi).

MnLOXs can be classified by their position of oxygenation of C₁₈ fatty acids into three categories. Gg-MnLOX in the first group oxygenates 18:2n-6 to 13R-hydroperoxy-9Z,11E-octadecadienoic acid (HPODE) and 11S-HPODE (11S-hydroperoxy-9Z,12Z-dienoic acid) in a ratio of 4:1 (13). Magnaporthe oryzae (Mo)- and Ms-MnLOX in the second category oxygenate 18:2n-6 to mainly 9S- and 11S-HPODE, and both catalyze the sequential oxygenation of 18:3n-3 to 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid (9S,16S-DiHPOTrE). The only enzyme of the third group is Fo-MnLOX, which oxygenates 18:2n-6 to the opposite stereoisomers, 13S- and 11R-HPODE. This also occurs by suprafacial hydrogen abstraction and oxygenation (9), which appears to be the catalytic hallmark of virtually all MnLOXs and is the main catalytic difference to iron lipoxygenase (FeLOX) (9–11, 13).

The 3D structure of Mo-MnLOX was reported recently (7). The sequence identity between Gg- and Mo-MnLOX is 56% (see Fig. 1A). Gg-MnLOX has been extensively studied since 1998 as the prototype MnLOX (12–15). The enzyme has been characterized by site-directed mutagenesis of residues, which were chosen from sequence homology to FeLOXs with known 3D structures or from sequence differences between MnLOXs with dissimilar catalytic activities (9–11, 16–19). The three dimensional structure of Gg-MnLOX might provide additional information of these tentative key residues and their importance for the catalytic activities of Gg-MnLOX and related MnLOXs (9–11, 16, 19, 20).

The present study had three major goals. The first goal was to solve the crystal structure of Gg-MnLOX from the data obtained during the first crystallographic analysis (21). The second goal was to compare the active sites of Gg- and Mo-MnLOX and a model of the active site of Fo-MnLOX to identify amino acids of catalytic importance. The third goal was to study hydrogen abstraction at the n-5 and n-8 positions of long chain fatty acids and hydroperoxides at the n-10 position by Gg-, Mo-, and Fo-MnLOX to find catalytic differences and, if possible, their relation to structural elements at the active sites. We also report the first crystal structure of a fungal adhesion protein with the same fold as collagen adhesins of Staphylococcus aureus and other Gram-positive bacteria.

MATERIALS AND METHODS

Materials

Fatty acids were from Larodan or VWR/Merck, and were stored at −20°C. The 11(R/S)- and 11S-HETE were from Cayman. Hydroperoxides of 18:2n-6 and 18:3n-3 were prepared as described (11, 19). Morpheus protein crystallization screen was from Molecular Dimensions. Expression of Mo- and Fo-MnLOX in Pichia pastoris (strain X-33) with the pPICZαA expression vector (Invitrogen) was performed as described (9, 11, 16). The secreted enzymes were purified by hydrophobic interaction chromatography and gel filtration as described (9, 11).

Enzyme expression and purification

The Gg-MnLOX precursor consists of 618 amino acids, including a predicted secretion signal of 16 amino acids (GenBank identification number (ID) AAK81882). Gg-MnLOX without the secretion signal was expressed (602 amino acids) and secreted by P. pastoris using pPICZαA with the yeast α-secretion signal (pPICZαA_MnLOX_602) (16). Fermentation was carried out in a 10 l bioreactor with 6 l of buffered minimal methanol medium for 4–5 days, as described (21). The medium was harvested, 136 g (NH₄)₂SO₄ was added per liter, the medium was adjusted with 10 M KOH to pH 6.8, which was followed by centrifugation or filtration. The medium was then used immediately for enzyme purification or stored at −80°C.

Gg-MnLOX was purified as described (21). Aliquots of the supernatant solution were captured by hydrophobic interaction chromatography (Phenyl Sepharose CL-4B) in 25 mM KHPO₄ buffer (pH 6.8):1.0 M (NH₄)₂SO₄. The columns were washed with the same buffer, and absorbed proteins were eluted with 25 mM KHPO₄ buffer (pH 7.0). The peak fractions were combined, concentrated (1–2 ml) by diafiltration, and deglycosylated in 0.1 M Na acetate:20 mM ZnCl₂ with α-mannosidase in a ratio of 1:100 (w/w) (21°C overnight). The reaction mixture was loaded on a gel filtration column (Superdex 200 HiLoad 16/60) in 25 mM Tris-HCl (pH 7.5):150 mM NaCl and eluted at 0.5 ml/min (ÄKTA FPLC; 21°C). The fractions with LOX activities were pooled, concentrated by diafiltration, and washed with 25 mM Tris-HCl (pH 7.5).

MALDI-TOF/TOF analysis

SDS-PAGE analysis revealed that the purified sample after gel filtration contained two dominating bands, an unknown protein of ∼23 kDa and a protein of ∼70 kDa (Gg-MnLOX after deglycosylation). They were identified by tryptic digestion and peptide mapping by MALDI-TOF/TOF analysis (Bruker Ultraflex). The Mascot search of the peptide map identified Gg-MnLOX in the 70 kDa band, but did not identify the unknown protein. MS/MS analysis of the two dominant peaks in the MS spectrum in the peptide map of the unknown protein identified two peptides, which contained residues 20-32 and 216-233 of a hypothetical protein, zonadhesin, of Komagataella phaffii (GenBank ID CCA40153). K. phaffii is identical to the strain of P. pastoris commonly used in gene expression studies (22). The peptide with residues 20-32 was identified as formed by tryptic cleavage at one position and thus contained the N-terminal end.

Enzyme assay

LOX activity was measured on a dual beam spectrophotometer (Shimadzu UV-2101PC). Enzyme was mixed with 100 μM of fatty acids in 0.1 M NaBO₃ (pH 9.0; at 22°C) and the UV-absorbance was followed at 235 or 237 nm or by repetitive UV spectra between 200 and 300 nm. The 9-HPOTrE (40–50 μM) (or the methyl ester) was incubated in the same way. K_m for Mo-MnLOX with 2.7–64 μM 9S-HPOTrE was estimated in triplicates (270 nm). Products were extracted on a cartridge of octadecyl silica (SepPak/C₁₈) and hydroperoxides were reduced to alcohols with triphenylphosphine before analysis (11).

Crystallization and X-ray diffraction data collection

Crystallization of Gg-MnLOX was set up using the sitting-drop vapor diffusion method at 20°C. The drops were prepared by mixing protein solution containing 20 mg/ml of protein with an equal amount of reservoir solution of the Morpheus screen in a 96-well plate (21). Data were collected with a Pilatus detector at wavelength 0.976 Å at 100 K on beam line ID29 at the European Synchrotron Radiation Facility, Grenoble, France. The data were processed with XDS (23) and iMosflm (24). The integrated data (iMosflm) were then scaled using SCALA in the CCP4 package (25). A set of 5% of the reflections was set aside and used to calculate the quality factor R_free (25). Statistics of data collection and processing are presented in Table 1.

TABLE 1.

Summary of data collection, processing, phasing, and structure refinement statistics

Data collection and processing
Beamline	ESRFa ID29
Detector	Pilatus 6M
Wavelength Å	0.976
Oscillation range	0.15°
Number of images	1,127
Space group	C2
Cell parameters a, b, c (Å); β (°)	226.6, 50.6, 177.9; 91.7
Resolution range (Å)	43.2–2.60
Number of observed reflections	160,569
Number of unique reflections	55,058
Average multiplicity	2.9
Completeness (%)	97.8 (98.7)
Rmerge (%)	0.095 (0.367)
Rmeas (%)	0.134 (0.517)
I/σ(I)	7.6 (2.4)
Refinement
Resolution used in refinement	43.2–2.6
Reflections in working/test set	60,262/3,049
Rwork/Rfree factor (%)	19.5/24.5
Molecules in asymmetric unit	2 lipoxygenases and 1 zonadhesin20-399
Number of atoms	11,543
Protein atoms	11,097
Manganese	2
Water molecules	222
Wilson B-factor	27.7
Average atomic B-factors (Å2)
Overall	27.0
Protein	26.6
rms (bonds)	0.014 Å
rms (angles)	1.36°

Statistics for the highest resolution shell are shown in parentheses.

^aESRF, European Synchrotron Radiation Facility, Grenoble, France.

Structure determination of Gg-MnLOX and zonadhesin

The structure of Gg-MnLOX was solved by molecular replacement with Phaser (26, 27) using molecule A of the Mo-MnLOX structure as a search model [Protein Data Bank (PDB) entry: 5FNO (7)]. There are two Gg-MnLOX molecules and one zonadhesin molecule in the asymmetric unit without any noncrystallographic symmetry present. Phenix.autobuild was able to construct the majority of the Gg-MnLOX molecules and main chain traces of the zonadhesin molecule. Further model building was performed in Coot (28). Structure refinements were performed with phenix.refine (29) to R_work of 0.208 and R_free of 0.245, respectively (Table 1).

Structure analysis

Sequences were aligned with the ClustalW program. Structure comparison and superpositioning were performed with PyMOL (PyMOL molecular graphics system, version 1.7.4, Schrödinger, LLC) and Coot (28), and the former was used to prepare figures. The phylogenetic tree was constructed with MEGA6 with bootstrap tests of the nodes as described (30). Mascot analysis was used to construct peptide maps as described (http://www.matrixscience.com). Swiss-Model was used to generate two homology models of Fo-MnLOX with Mo-MnLOX and Gg-MnLOX as templates, respectively (31). The 18:2n-6 was docked to Gg-MnLOX and Mo-MnLOX with the SwissDock program (32), and the mol2 file for docking of 18:2n-6 was generated from the ligand library in SwissDock.

LC-MS analysis

Reversed-phase (RP)-HPLC with MS/MS analysis was performed with a Surveyor MS pump (ThermoFisher) and an octadecyl silica column (5 μm; 2.0 × 150 mm; Phenomenex), which was eluted at 0.3 ml/min with methanol:water:acetic acid, 750:250:0.05 for separation of hydroxy fatty acids and 650:350:0.05 for separation of dihydroxy fatty acids. The effluent was subject to electrospray ionization in a linear ion trap mass spectrometer (LTQ, ThermoFisher). The heated transfer capillary was set at 315°C, the ion isolation width at 1.5 atomic mass units, the collision energy at 35 (arbitrary scale), and the tube lens at about −110 V. Prostaglandin F_1α was infused for tuning. Samples were injected by an auto-injector (Surveyor autosampler plus; Thermo).

Normal-phase (NP)-HPLC with MS/MS analysis was performed with a manual injector (Rheodyne 7510), a silicic acid column (5 μm; 250 × 2 mm, Reprosil; Dr. Maisch), eluted at 0.5 ml/min with hexane:isopropyl alcohol:acetic acid, 98:2:0.05 (Constametric 3200 pump, LDC/MiltonRoy). The effluent was combined with isopropyl alcohol:water, 3:2 (0.3 ml/min) from a second pump (Surveyor MS pump). The combined effluents were introduced by electrospray ionization into the ion trap mass spectrometer above. Stereoisomers of HETE were resolved by chiral-phase (CP)-HPLC on Reprosil Chiral-AM (5 μm, 250 × 2 mm; Dr. Maisch), eluted at 0.2 ml/min with hexane:methanol:­acetic acid, 96:4:0.025, and mixed with isopropyl alcohol:water, 3:2 (0.15 ml/min).

Miscellaneous

Protein concentration was estimated by UV absorption at 280 nm. SDS-PAGE was performed as described (11). Diazomethane was used for methylation.

RESULTS

Crystallization and structure determination

Commercial crystallization screens were used to identify suitable crystallization conditions for Gg-MnLOX. The best crystals appeared as described (21) in solution A3 from Morpheus Screen containing 0.1 M MES-imidazole pH 6.5, 20% (v/v) glycerol, 10% (w/v) PEG-4000, 0.03 M MgCl₂, and 0.03 M CaCl₂.

A data set with more than 1,100 consecutive images with an oscillation range of 0.15 degrees and exposure time of 0.043 s was collected to a resolution of 2.6 Å from a single crystal, as described (21). The crystal belongs to the space group C2 with unit-cell parameters: a = 226.6 Å, b = 50.6 Å, c = 177.92 Å, and β = 91.70° (21).

There are two molecules of Gg-MnLOX and one molecule with residues 20-339 of zonadhesin (zonadhesin_20-339) (GenBank ID CCA40153) in the asymmetric unit with a Matthews coefficient of 3.2 ųDa⁻¹ and a solvent content of 62%. We previously reported that there could be either two or three molecules of Gg-MnLOX in the asymmetric unit, as judged from the self-rotation function (26), and this has now been clarified. The third molecule, zonadhesin, consists of 587 residues (33), but the amino acids 1-19 and 340-587 could not be detected. Loss of the former as a secretion signal of 19 amino acids was confirmed by MALDI-TOF/TOF analysis of the tryptic peptide, which was formed by single cleavage, and contained residues 20-32 as described above. This crystallized protein was designated zonadhesin_20-339.

The sequence identity between Mo-MnLOX and Gg-MnLOX is 56%, and this allowed us to solve the structure by molecular replacement. The electron density was poor on a few locations. Gg-MnLOX could be built from Gln-46 to Val-618 with gaps between Ala-424 and Asp-427 and between Ala-510 and Leu-519. Zonadhesin was built from Ala-20 to Val-339 with gaps between Ala-233 and Ser-241 and between Ala-301 and Asp-307. The atomic coordinates and structure factors have been deposited in the PDB (PDB entry: 5FX8).

Gg-MnLOX contains six predicted Asn glycosylation sites (NetNGlyc 1.0 server) and five of them (Asn-60, -91, -106, -116, and -157) contained one to three N-acetylglucosamine residues. The sixth position, Asn-513, is located between Ala-510 and Leu-519 in the gap with poor electronic density.

Overall structure of zonadhesin and its interaction with Gg-MnLOX

The 3D structure of zonadhesin_20-339 is shown as a cartoon in the asymmetric unit in Fig. 2A. The molecule has two domains (N₁ and N₂), which are dominated with β-strands. Each domain contains two anti-parallel β-sheets forming one β-sandwich and the N-terminal domain has a short α-helix (Glu-135 to Thr-138) connecting two β-strands. The fold of the two domains with β-sandwiches can be described as IgG-like (see below). The sequence of the missing C-terminal domain with 248 residues lists 64 Thr/Ser residues and 68 Gly/Ala/Val residues in characteristic repeat of adhesins (34, 35).

Fig. 2.

Overview of the asymmetric unit and the interaction between zonadhesin and surrounding molecules in crystal packing. A: Carton representation of the asymmetric unit with zonadhesin in rainbow colors (N-terminal, blue; C-terminal, red; chain, U) and chains A and B of Gg-MnLOX (purple and green, respectively). B: Overview of the crystal packing of four molecules of Gg-MnLOX (two chains A in purple, two chains B in green) and two molecules of zonadhesin (U chains, colored in rainbow and in gray).

The interaction at the interface of zonadhesin_20-339 and Gg-MnLOX appeared to be partly electrostatic, as judged from the surface charge of the molecules at the contact area. The PDBePISA program (http://www.ebi.ac.uk) identified four salt bridges and fourteen hydrogen bonds between Gg-MnLOX (chain A) and zonadhesin_20-339 (chain U) at an interface area of 686 Ų. The predicted pI of zonadhesin_20-339 is 3.6, whereas the isoelectric point of Gg-MnLOX is estimated to be 9.7 (12). The crystal packing of two A and B chains of Gg-MnLOX with two molecules of zonadhesin_20-339 is shown in Fig. 2B. The latter did not bind near the entrances of the substrate channels. Zonadhesin_20-239 can be removed by cation exchange chromatography (16), and we found that the catalytic activity of Gg-MnLOX with and without zonadhesin_20-239 appeared to be similar.

Overall structure of Gg-MnLOX and a comparison with Mo-MnLOX

An illustration of the overall structure of Gg-MnLOX is presented in Fig. 3A. Gg-MnLOX lacks the β-barrel domain with homology to the polycystin-1-lipoxygenase-α-toxin domain of FeLOX. Gg-MnLOX and Mo-MnLOX share the same fold as illustrated by superimposing their structures (Fig. 3B). The C_α carbons of the metal ligands of Gg- and Mo-MnLOX align with a root-mean-square (rms) deviation of 0.34 Å. The geometry of the metal ligands is also conserved (Fig. 3C). The overall rms deviation of Gg- and Mo-MnLOX is 0.51 Å.

Fig. 3.

Overall structure of Gg-MnLOX and a comparison with Mo-MnLOX. A: Cartoon illustration of the overall structure of Gg-MnLOX (PDB entry: 5FX8) in two directions 90° away from each other. The protein is colored in rainbow spectrum with the N terminal in blue and C terminal in red. The broken helix covering the active site with the catalytic manganese (gray) is colored in light green. B: Cartoon illustration of Gg-MnLOX (rainbow) superimposed with the cartoon illustration of Mo-MnLOX (gray; PDB entry: 5FNO) with an rms deviation of 0.51 Å. C: The catalytic manganese (gray sphere) is coordinated by His-290, His-294, His-469, Asn-482, and Val-618 of Gg-MnLOX (green), and the identical metal ligands of Mo-MnLOX (gray) are superimposed with an alignment of the C_α carbons with an rms deviation of 0.34 Å. The catalytic water is marked by a red sphere, and the side chains are shown except for the main chain of Val-618.

His-290, His-294, His-478, Asn-482, the carboxyl oxygen of Val-618, and a water molecule coordinate the catalytic manganese (Fig. 3C). Four of the five manganese ligands are essential for catalysis (16). His-290 and His-294 are separated by three residues in Gg-MnLOX,⁴ and the corresponding His-284 and His-289 residues of Mo-MnLOX⁵ by an additional residue, Pro-288. The electron density over this area, from Tyr-289 to Glu-298, of Gg-MnLOX is shown in Fig. 4A. This region of the structure is superimposed with the corresponding region of Mo-MnLOX (Phe-283 to Glu-298) in Fig. 4B. The two pairs of His residues are well aligned, likely due to the compactness of the inserted Pro residue. Interestingly, the Pro-288 residue is not conserved in other MnLOX. A Thr residue is found at this position of Fo-MnLOX and often a Gly residue in FeLOX, but insertion of a Thr or Gly residue between His-290 and His-294 of Gg-MnLOX at the corresponding position of Pro-288 inactivated the enzyme (19). A summary of the effects of replacement of residues in the active site of Gg-MnLOX is shown in Table 2.

Fig. 4.

Overview of the manganese ligands His-290, His 294, and Asn-472 of Gg-MnLOX and a comparison with Mo-MnLOX. A: An unbiased 2F_o-F_c electron density map of the sequence of ten amino acid residues covering His-290 and His-294 at the contour level of 1 σ. B: The figure shows the three dimensional structure with sticks of the sequence in (A) (colored light green) and an overlay of the corresponding sequence of Mo-MnLOX (colored light blue). C: Superpositioning of the loop with Asn-482 of Gg-MnLOX with the corresponding loop with Asn-473 of Mo-MnLOX, and the α-helix of 8R-LOX (PDB entry: 4QWT). Gg-MnLOX, light green; Mo-MnLOX, light blue; 8R-LOX, light gray.

TABLE 2.

Overview of site directed mutagenesis of Gg-, Mo-, and Fo-MnLOX, major catalytic effects, and possible structural correlations

Residue	Mutation	Catalytic Effect	Location	Interpretation
Gg-MnLOX
His-290	Gln	Loss of activity/Mn	Metal center	Mn liganda
Insertion	Thr-293	Loss of activity	Active site	Mn coordination
	Gly-293	Loss of activity		Mn coordination
His-294	Gln	Loss of activity/Mn	Metal center	Mn liganda
Gly-332	Ala	Epoxyalcoholsb	Active site	Constriction/reduced O2
	Val	Loss of activity		Constriction/reduced O2
Leu-336	Ala	Oxygenation of C-9c	Active site	Positioning/O2 access
	Gly	Oxygenation of C-9c		Positioning/O2 access
Phe-337	Ile	13R to 13S chiralityd	Active site	Shielding O2
Phe-347	Leu	Oxygenation at C-9c	Channel end	Positioninge
	Val	Oxygenation at C-9c		Positioninge
	Ala	Oxygenation at C-9c		Positioninge
His-478	Gln	Loss of activity/Mn	Metal center	Mn ligand
His-479	Gln	Retained activity	Metal center	Nonessentiala
Asn-482	Gln	Retained activity	Metal center	Nonessentiala
	Leu	Retained activity		Nonessentiala
Gln-483	Asn	Retained activity	Active site	Nonessential
Val-618	Deletion	Loss of activity/Mn	Metal center	Mn liganda
	Ile	Retained activity		Carboxyl group essentiala
	Ala	Retained activity		Carboxyl group essentiala
Mo-MnLOXf
Arg-525	Ala	Loss of activityh	Entrance	Tethering
Phe-526	Leu	Loss of activityh	Active site	Positioning
Fo-MnLOXg
Ser-346	Phe	11R to 11S chirality	Channel end	Positioning
Leu-530	Arg	Retained activity	Entrance	Nonessential

^aData from (16).

^bThis mutation increases the biosynthesis of epoxyalcohols, but has little effect on the regiospecificity (17).

^cThese mutations increase the relative oxygenation at C-9 of 18:2n-6 (19).

^dThis mutation shifts the direction of oxygen insertion but retains the steric abstraction of the hydrogen (19).

^eThis residue aligns with the Sloane determinant at the bottom of the substrate channel (10).

^fData from (7).

^gData from (9).

^hOxygenation of fatty acids was almost abolished, but 9-HPOTrE was oxygenated at C-16 (7).

A small loop connecting α-helix 16 (α16) and α17 close to the active site harbors the metal ligand Asn-482_. This loop may bring Asn-482 close to the catalytic metal (Table 3). This loop is also present in Mo-MnLOX (Fig. 4C), but corresponds to an α-helix in 8R-LOX (7, 36). The oxygen of Gln-287 forms tentative hydrogen bonds to the nitrogen of Asn-482 with a distance of 2.9 Å. His-290, Val-618, and Asn-482 may form hydrogen bonds with the catalytic water (2.6, 3.0, and 3.2 Å, respectively).

TABLE 3.

A comparison of the distance between the corresponding metal ligands and Gg- and Mo-MnLOX with CspLOX2 and sLOX-1

Metal Ligands	Distances
	Gg-MnLOX	Mo-MnLOXa	CspLOX2b	sLOX-1c
His-290 (NE2)	2.4	2.6	2.5	2.23
His-294 (NE2)	2.1	2.2	2.5	2.26
His-478 (NE2)	2.2	2.1	2.5	2.21
Asn-482 (OD1)	2.3	2.9	2.9	3.05
Val-618 (OXT)	1.9	2.1	2.5b	2.40c

^aData from (7).

^bIle-668 is the C-terminal residue of CspLOX2 (6).

^cIle-839 is the C-terminal residue of soybean lipoxygenase-1 (sLOX-1) (8).

The α2 helix extends along one side of the protein from residue Glu-87 to Ser-124, and four of its residues are found at the entrance to the active site (Trp-100, Ala-104, Thr-108, and Tyr-112). The sequence from α9 to the end of α10 is described as the arched helix (1), and it contains a series of important residues, e.g., Ile-328, which clamps the fatty acid against the catalytic base for hydrogen abstraction (1), the Gly-Ala switch/Coffa-Brash determinant Gly-332 in the wall of the active site (37), Leu-336, Phe-337, and the Sloane determinant Phe-347 in the bottom of the active site (38). Trp-343, Leu-535 and Met-288 also build this part of the channel. Arg-538 at the end of α18 tethers the carboxyl group of fatty acids, and the side chain of the next residue, Phe-539, is positioned close to Leu-336 and Phe-337 in the active site.

The “clamp” residue Leu-336, the catalytic water, manganese, and His-474 align close to a straight line and C-11 of 18:2n-6 is positioned here for hydrogen abstraction. Gg- and Mo-MnLOX catalyze suprafacial hydrogen abstraction and oxygenation, which implies that O₂ can access the pentadienyl radical from the same side as the catalytic metal. The other side is likely shielded by Phe-337 of Gg-MnLOX during oxygenation of C-13 and C-11. The aligned sequences of Gg-, Mo-, and Fo-MnLOX with marked residues from known positions of the 3D structures are shown in Fig. 5 to facilitate a comparison of the enzymes and the numbering of amino acids in critical positions. Four residues of Mo- and Fo-MnLOX have also been investigated by site-directed mutagenesis (Table 2).

Fig. 5.

Sequence alignment of Gg-, Mo-, and Fo-MnLOX. ClustalW aligned the sequences, the figure was prepared with ESPript (http://espript.ibcp.fr) and the numbers on top of the sequences apply to the Gg-MnLOX sequence (AAK81882). The secondary structures of Gg-MnLOX are shown above the alignments. Important residues, which were deduced from the 3D structure, are marked under the alignment as follows: Residues at the entrance of the substrate channel are marked by a blue ring, the Coffa-Brash and the Sloane determinants with blue hash tags (#). Important residues, which are discussed in the text and figures, are denoted in the same way with green (conserved residues) or red (variable residues) angles (^). The metal ligands are marked by an asterisk (*).

The substrate channel of Gg-MnLOX contains two conspicuous side pockets near the catalytic center (Fig. 6A, B). The side pockets are delineated by a series of residues, e.g., Leu-77, Met-288, Val-291, Thr-295, Ile-328, Phe-342, and Trp-343 (Figs. 5, 6). Some of them are replaced with larger or smaller hydrophobic residues in Mo-MnLOX (Ile-282, Ala-290, Val-323, Leu-337, and Phe-338) (Fig. 6C, D). The channel entrance and the proximal pocket appear to be slightly larger in Mo- than in Gg-MnLOX and the distal pocket larger in Gg-MnLOX. The proximal and distal side pockets near the catalytic center might be important for oxygenation at C-9 and C-13 of C₁₈ fatty acids, respectively. Oxygen likely enters by the same route as the fatty acid, as we could not identify a tentative oxygen channel of Gg-MnLOX.

Fig. 6.

Overview of the substrate channel of Gg-MnLOX with surrounding amino acid residues and a comparison with Mo-MnLOX. A: The substrate channel of Gg-MnLOX is viewed with the entrance to the right. Conserved residues in Gg-, Mo-, and Fo-MnLOX are marked green, and residues, which differ between the three lipoxygenases, are marked in red. B: The substrate channel in (A) is rotated 90° and viewed with the entrance opening away from the spectator. C, D: The figures show the substrate channel of Mo-MnLOX in the same directions as in (A) and (B), respectively. Manganese is shown as the violet circle in (B) and (D) and as a dark circle in (A) and (C). Marked in red are five residues of the substrate channel that differ from Gg-MnLOX: Ile-282, Ala-290, Val-323, Leu-337, and Phe-338.

We used docking of 18:2n-6 to estimate the substrate binding position in the active site. A model of 18:2n-6 in the active site of Mo-MnLOX yielded the best docking score based on energy minimization. The docked molecule fitted in the substrate channel with the 9Z double bond facing the proximal pocket, C-11 near the metal center, the 12Z double bond facing the distal pocket, and the carboxyl group at hydrogen bond distance to Arg-525. To compare the active site with the docked substrate, Gg-MnLOX and a model of Fo-MnLOX (see below) were superimposed onto Mo-MnLOX.

Four residues of α2 (Trp-100, Ala-104, Thr-108, and Tyr-112), two residues of α9 (Val-331 and Val-335), and Phe-342 delineate the entrance to the active site of Gg-MnLOX (Fig. 7A). Arg-538 is positioned in the substrate channel and could tether the carboxyl group of fatty acids in the same way as Arg-525 of Mo-MnLOX (7). The entrance of the substrate channel could be less accommodating in Gg-MnLOX than in Mo-MnLOX due to the Phe-342 residue, which is replaced by a Leu residue in Mo-MnLOX (Fig. 7B). The entrance appears to be even smaller in the model of Fo-MnLOX (Fig. 7C) described below.

Fig. 7.

Overview of the entrances to the substrate channels. A: The figure shows the entrance to the active site of Gg-MnLOX and surrounding amino acids. B: Entrance to the active site of Mo-MnLOX. C: Model of the entrance to the active site of Fo-MnLOX. The variable amino acids are marked in red and the conserved are marked in green. Fo-MnLOX lacks an Arg residue at the entrance, replaced by a Leu-530 residue at the corresponding position of Arg residues of Gg- and Mo-MnLOX. The 18:2n-6 [linoleic acid (LA) in yellow] was docked into the active sites by the SwissDock program using Mo-MnLOX (PDB: 5FNO) as a target. All three models were superimposed in PyMol with 18:2n-6 for comparison.

Gly-332, Leu-336, Phe-539, Val-618, and Ile-328 of Gg-MnLOX are located near the catalytic metal below the entrance to the substrate channel, as shown in Fig. 8A. Replacement of Gly-332 (Gly-Ala switch/Coffa-Brash determinant) with larger hydrophobic residue (Ala, Val) likely narrows the channel and could reduce the access of oxygen and substrates to the active site (Fig. 8B, C). The effects of the Gly332Ala and Gly332Val replacements are summarized in Table 2.

Fig. 8.

Overview of the position of Gly-332 of Gg-MnLOX. A: The surface of Gly-332 in the orifice above the catalytic metal (not shown) is marked red. B: Replacement of Gly-332 with Ala (yellow) narrowed the orifice, which may explain the catalytic activity of the Gly332Ala mutant (Table 2). C: Replacement of Gly-332 with Val (blue) markedly narrowed the channel, and inactivated the enzyme (Table 2). In addition to Gly-332, Leu-336, Phe-539, Val-618, and Ile-328 delineate the orifice as indicated.

We conclude that the different catalytic properties of Gg- and Mo-MnLOX could be related to the volumes and the width of the substrate channel and the two side pockets.

Homology models of Fo-MnLOX

Fo-MnLOX shares about 55% sequence identity with Gg- and Mo-MnLOX (Fig. 1). Two models of Fo-MnLOX were generated using the PDB files of Gg- and Mo-MnLOX as templates (PDB entries: 5FX8, 5FNO). As expected, the two homology models were similar and showed an rms deviation of 0.5 Å for all atoms. Unexpectedly, both models showed a tentative oxygen channel to the active site and the side pockets of the two templates were hardly detected. The entrance to the substrate channel was relatively narrow as illustrated in Fig. 7C.

Lipoxygenation of 9S- and 9R-HPOTrE

The Arg525Leu mutant of Mo-MnLOX oxygenated 18:2n-6 insignificantly, but 9S-HPOTrE was oxygenated rapidly at C-16 (7). Whether 9S-HPOTrE is tethered by another Arg residue or metabolized by Gg- or Fo-MnLOX are unknown. Based on these considerations, we examined the mechanism of oxygenation of 9S-HPOTrE, the methyl ester, and the 9R stereoisomer.

Mo-MnLOX oxygenated 9S- and 9R-HPOTrE to 9S,16S-DiHPOTrE and 9R,16S-DiHPOTrE, respectively, which occurs by hydrogen abstraction at the n-5 position. The two diastereoisomers were separated by RP-HPLC (Fig. 9A). Both 9S- and 9R-HPOTrE were oxygenated at C-16 with stereo specificity (>95% S) (see inset in Fig. 9A). The 9-hydroperoxide was not essential as Mo-MnLOX oxygenated the alcohol, 9S-HOTrE, in the same way.

Fig. 9.

LC-MS/MS analysis of the lipoxygenation of 9-HPOTrE, 20:5n-3, and 20:4n-6. All products were analyzed after reduction of hydroperoxides to alcohols. A: RP-HPLC-MS/MS analysis of 9,16-dihydroxyoctadecatrienoic acid (DiHOTrE), which were formed separately from 9S- and 9R-HPOTrE by Mo-MnLOX and combined for analysis. The 9S,16S-DiHOTrE has the shortest retention time (11). The inset shows that 9R-HPOTrE is transformed by Mo-MnLOX to 9R,16S-HPOTrE as the main stereoisomer (>95%). B: Oxygenation of 20:5n-3 and 20:4n-6 (inset) by Gg-MnLOX. The 20:5n-3 was transformed to 15- and 13-HEPE as the two major metabolites, which were separated by NP-HPLC, and to small amounts of 11-HEPE. The inset shows that Gg-MnLOX oxygenates 20:4n-6 to 15-HETE and only to small amounts of 13-HETE and 11-HETE (NP-HPLC-MS/MS analysis) as end products. C: Oxygenation of 20:5n-3 and 20:4n-6 (inset) by Mo-MnLOX. RP-HPLC-MS/MS analysis of products formed from 20:5n-3 by Mo-MnLOX revealed three peaks with metabolites: 11,18-DiHEPE in peak “11,18”, 13-HEPE in peak “13”, and 15- and 11-HEPE in peak “11 and 15”. The inset shows NP-HPLC-MS/MS analysis of end products formed from 20:4n-6 by Mo-MnLOX: 15R-HETE, 13S-HETE, and 11S-HETE eluted as indicated. D: Oxygenation of 20:5n-3 by Fo-MnLOX. The 16- and 14-HEPE were formed as main products along with small amounts of 18-HEPE. The inset shows selective ion monitoring of the intensities of m/z 207 (14-HEPE, marked 14) and m/z 215 (18-HEPE; marked 18). E: Oxygenation of 20:4n-6 by Fo-MnLOX. The chromatogram shows NP-HPLC-MS/MS analysis and separation of 15-, 13-, and 11-HETE. F: CP-HPLC-MS/MS analysis of 13-HETE and 11-HETE formed by Fo-MnLOX. The R and S stereoisomers of 13- and 11-HETE eluted as indicated.

UV analysis showed that the methyl ester of 9S-HPOTrE was transformed by Mo-MnLOX to 9,16-DiHPOTrE with the development of the typical triene UV spectrum with λ_max at 270 nm and shoulders at 260 and 280 nm (11). K_m for the oxygenation of 9S-HPOTrE was estimated by UV analysis (270 nm) in triplicate to be ∼3 μM.

UV analysis showed that Fo-MnLOX transformed 9S-HPODE to a triene, which was identified by LC-MS as 9S,16S-DiHPOTrE (>95%). Gg-MnLOX did not oxygenate 9S-HPOTrE to a triene according to UV analysis, and this was confirmed by LC-MS analysis of products after prolonged incubation. The oxygenation of 9-HPOTrE by Mo- and Fo-MnLOX, and not by Gg-MnLOX, could be due to binding of 9-HPOTrE to Phe-342 or other hydrophobic residues in unproductive configurations.

Oxygenation of long chain fatty acids by Mo-, Fo-, and Gg-MnLOX

Methyl esters of fatty acids are not oxygenated by Gg-MnLOX, and the carboxyl group of C₁₈ fatty acids are likely tethered to the Arg residues at the entrance of the substrate channel (7). The number of double bonds reduces the flexibility of fatty acids, and a comparison of the oxygenation of 20:4n-6 and 20:5n-3 may therefore illustrate the conformation of the active sites.

The 20:5n-3 was oxygenated by Gg-MnLOX to 13- and 15-hydroperoxyeicosapentaenoic acid (HPEPE) in almost equal amounts, and to small amounts of 10-HPEPE (Fig. 9B). The 13-HPEPE is formed by hydrogen abstraction and oxygenation of the bis-allylic C-13, whereas 15-HPEPE is formed by oxygenation of C-15 with the typical shift in the position of the double bond (14Z→13E). The n-3 double bond apparently positioned C-13 and C-15 for oxygen insertion to the same extent. This is in contrast to the oxygenation of 20:4n-6 (inset in Fig. 9B). The latter was oxygenated to 15R-hydroperoxyeicosatetraenoic acid (HPETE) as the main metabolite and only to small amounts of 13S- and 11S-HPETE as judged from NP- and CP-HPLC-MS/MS analysis (39).

Mo-MnLOX also oxygenated 20:5n-3 and 20:4n-6 by hydrogen abstraction at C-13. Mo-MnLOX oxygenated 20:5n-3 to 11-, 13-, and 15-HPEPE. The 11-HPEPE was sequentially oxygenated to 11,18-DiHPEPE in analogy with the sequential lipoxygenation of 18:3n-3 and 20:3n-3 by hydrogen abstraction at first the n-8 and then the n-5 positions (Fig. 9C) (7). The 20:4n-6 was oxygenated to 13-HPETE, 11-HPETE, and 15-HPETE. The 13-HPETE was subject to β-fragmentation after prolonged incubation and decreased (inset in Fig. 9C). Steric analysis by CP-HPLC showed that 13S-HETE eluted as a single peak along with 15R- and 11S-HETE.

Fo-MnLOX transformed 20:5n-3 to 14- and 16-HPEPE and to small amounts of 18-HPEPE, apparently by hydrogen abstraction at the n-5 position (Fig. 9D). The MS/MS spectrum of 14-hydroxyeicosapentaenoic acid (HEPE) (m/z 317→full scan) showed an important signal at m/z 235 (317-82; loss of H₂C=CH-CH=CH-CH₂-CH₃), 207 (235-28; loss of CO) and m/z 163 (207-44; loss of CO₂) [see the fragmentation of 11-HEPE with signals at m/z 195 (235-40) and m/z 167 (207-40)]. The spectrum of 16-HEPE showed signals, among other things, at m/z 233 (317-84; loss of HCO-CH=CH-CH₂-CH₃), m/z 217 (317-100; loss of CO₂ and H₂C=CH-CH₂-CH₃), and m/z 189 (233-44). As expected, 16-HEPE was hydrolyzed by 0.001 M HCl to 14- and 18-HEPE [see (13)]. The 18-HEPE showed charac­teristic signals at m/z 259 (317-58; loss of HCO-CH₂-CH₃) and m/z 215 (317-102; loss of CO₂ and HCO-CH₂-CH₃). Fo-MnLOX thus oxygenates 20:5n-3 by hydrogen abstraction at the n-5 position in contrast to hydrogen abstraction at the n-8 position by Gg- and Mo-MnLOX. Hydrogen abstraction at the n-5 position with oxygenation at the n-7 position is also catalyzed by FeLOX of the cyanobacterium Acaryochloris marina (40), but oxygenation at the n-5 and n-3 positions by Fo-MnLOX appears to be unprecedented.

Fo-MnLOX also oxygenated 20:4n-6 to 13-HPETE as a major metabolite along with 11- and 15-HPETE (Fig. 9E). The 13S and 13R stereoisomers of 13-HETE were formed in a ratio of 4:1 (Fig. 9F, top). This was unexpected, as Fo-MnLOX oxygenates 18:2n-6 to 11R-HPODE (9). Hydrogen abstraction at the n-8 position of 20:4n-6 and 18:2n-6 is followed by oxygen insertion at this position in different orientations. Fo-MnLOX also formed 15- and 11-HPETE. The 15-HPETE was almost racemic (55% R), but 11-HETE was formed with stereo selectivity (>95% S; Fig. 9F, bottom).

The 22:5n-6 was oxygenated slowly compared with 20:4n-6, but the products illustrate the catalytic differences of Gg- and Mo-MnLOX. Both enzymes abstracted the hydrogen at C-15 (n-8), but Gg-MnLOX formed the hydroperoxide at C-17 (n-6) as the main metabolite, whereas Mo-MnLOX oxygenated C-17 and C-13 (n-10) to the same extent (7). Fo-MnLOX was also mainly oxygenated at C-17.

We conclude that there are productive configurations in the active sites, which allow hydrogen abstraction at the n-8 and n-5 positions of 20:5n-3, the n-5 position of 11-HPEPE/ 9-HPOTrE, and the n-8 position of 20:4n-6 and 22:5n-6. Hydrogen abstraction at the n-5 positions could be related to the confined configurations of the substrate channels of Mo- and Fo-MnLOX in relation to Gg-MnLOX discussed above.

DISCUSSION

We report the three dimensional structures of Gg-MnLOX and zonadhesin_20-339 at a resolution of 2.6 Å. The structure of Gg-MnLOX revealed that the geometry of the metal ligands was conserved in comparison with Mo-MnLOX, but demonstrated volume differences in the substrate channels. Many assumptions of the positions and functions of amino acid residues of Gg-MnLOX, which have been subject to site-directed mutagenesis (Table 2), can now be analyzed with greater confidence.

Gg-MnLOX preferentially oxygenates C-13/C-11, Mo-MnLOX C-9/C-11, and Fo-MnLOX C-11/C-13 of 18:2n-6. The U-shaped substrate channels differ by the size of two pockets on both sides of the catalytic center (Fig. 10). The volume of the proximal pocket (marked I in Fig. 10) is the largest in Mo-MnLOX, slightly smaller in Gg-MnLOX, and hardly noticeable in the model of Fo-MnLOX. The distal side pocket (marked II) decreases in volume from Gg- to Fo-MnLOX. The model suggests that the distal pocket of the latter could be connected to the surface by a tentative oxygen channel. Overall, the interior of the substrate channel of Gg-MnLOX is the widest and the deduced channel of Fo-MnLOX is presumably the most restricted of the three enzymes.

Fig. 10.

Comparison of the active sites of Gg-, Mo-, and Fo-MnLOX. A: Active site of Gg-MnLOX. B: Active site of Mo-MnLOX. C, D: The active site in two directions of a homology model of Fo-MnLOX; (C) has the same view as (A) and (B) and (D) is rotated 45° away to show the putative oxygen channel. All three models were superimposed in PyMol for comparison. Residues that are important for the shape of the substrate channels are shown in blue. The proximal and distal pockets near the catalytic metal are marked I and II, respectively, in (A) and (B). Red arrows in (C) and (D) mark the putative oxygen channel. Linoleic acid (LA, shown in yellow) was docked into the active sites by the SwissDock program using Mo-MnLOX (PDB: 5FNO) as a target.

Phe-347 is positioned at the far end of the active site, known as the position of the Sloane determinant (38) (Fig. 6). The importance of Phe-347 for the regiospecificity of Gg-MnLOX is well-documented. First, Phe347Leu, Phe347Val, and Phe347Ala increased sequentially the oxygenation of 18:2n-6 at C-9 (10) (Table 2). Second, Ms-MnLOX with Leu-350 in this position mainly oxygenated C-9 of 18:2n-6 (9). Ser-346 is found in this position of Fo-MnLOX (Fig. 5) and the Ser346Phe mutation changed the chirality of the products (Table 2), presumably by repositioning of the substrate (9).

Leu-336 and Phe-337 of Gg-MnLOX appear to position the substrate near Mn²⁺OH₂ for suprafacial hydrogen abstraction and oxygenation (Table 2). Phe residues are conserved in this position of MnLOXs, whereas Ile or Leu is present in FeLOXs (19). Phe337Ile retained abstraction of the proS hydrogen at C-11 of 18:2n-6, but altered the oxygenation by Gg-MnLOX from biosynthesis of 13R- to 13S-HPODE (19). This result and the three dimensional structure suggest that Phe-337 could shield one side the n-6 double bond for oxygenation, which could be essential for the suprafacial oxygenation mechanism. The crystal structure of Gg-MnLOX with 18:2n-6 in the active site will be needed to confirm this mechanism.

Gly-332 is located at the position known as the Gly-Ala switch (1, 37). Gly or Ala at this position of many FeLOX can shift the oxygenation of 18:2n-6 between C-9 and C-13 (37). Gly-332 is located in the wall of the active site of Gg-MnLOX. The fact that Gly332Ala increased the hydroperoxide isomerase activities and Gly332Val inhibited catalysis can now be explained by space restrictions in the active site (17) (Fig. 8). Replacement of the corresponding Ala-451 of eLOX-3 with Gly reduced the prominent hydroperoxide isomerase activities, presumably by increasing oxygen access to the active site (41). Ala-451 of eLOX3 and Gly-332 of Gg-MnLOX illustrate a second effect of the Gly-Ala switch.

The 9S-HPOTrE is not oxygenated at C-16 by Gg-MnLOX. This is the most striking catalytic difference to Mo- and Fo-MnLOX. This lipoxygenation is unique in several aspects. The Arg-525 residue of Mo-MnLOX is not required (7), the methyl ester of 9S-HPOTrE is oxygenated, and the chain length is not critical (Fig. 9). Tethering of the carboxyl group of 9S-HPOTrE by other positively charged residues can therefore be excluded. Both 9S- and 9R-HPOTrE are oxygenated with S stereo configuration at C-16 (Fig. 9A). This suggests that 9-HPOTrE is positioned with C-14 (n-5) of the 12Z,15Z-pentadiene at the catalytic base for hydrogen abstraction. The lack of oxygenation of 9-HPOTrE by Gg-MnLOX could be due to binding of 9-HPOTrE in an unproductive configuration, apparently caused by differences in the substrate channels of Gg- and Mo-MnLOX (Figs. 6, 7, 10). The homology model of Fo-MnLOX suggests a relatively narrow substrate channel. This enzyme nevertheless oxygenated 9S-HPOTrE by hydrogen abstraction at the n-5 position. Fo-MnLOX lacks an Arg residue at the entrance of the substrate channel for tethering of carboxyl groups of fatty acids (Figs. 5, 7C). The oxygenation of fatty acids by hydrogen abstraction at the n-8 or n-5 positions and 9-HPOTrE suggest that the substrates enter the active site of Fo-MnLOX “tail first” as indicated in Figs. 7 and 10.

The metal ligands, His-290 and His-294, of Gg-MnLOX are located along a standard helix. Mo-MnLOX harbors an additional Pro-288 residue between the corresponding His-284 and His-289 residues (Figs. 4, 5). This nevertheless results in an almost identical orientation of the two pairs of His residues.

The ligand, Asn-482, of Gg-MnLOX is situated on a short loop 2.8 Å away from manganese and not on a helix in analogy with Asn-473 of Mo-MnLOX (7), and these ligands could therefore be relatively mobile. The mutants, Asn482Leu and Asn482Gln, of Gg-MnLOX retained catalytic activities, whereas the other metal ligands are required (Table 2) (16).

The 11-HPODE is subject to rapid enzymatic β-fragmentation by Gg- and Mo-MnLOX (11, 13). This also occurs nonenzymatically by Mn³⁺ in methanol, but not by Fe³⁺ (42, 43). FeLOX catalyze β-fragmentation at an insignificant rate with one exception, the 9R-LOX of Cyanothece (CspLOX2) (18, 44). Enzymatic β-fragmentation likely requires positioning of the hydroperoxide group near the catalytic center and steric factors are therefore of obvious importance (42, 43). In addition, the metal ligands may adjust the redox potentials of protein-bound iron and manganese to support β-fragmentation and lipoxygenation. The redox potentials of Mn²⁺/Mn³⁺ and Fe²⁺/Fe³⁺ differ by a factor of two, and Mn-substituted FeLOX lose their catalytic activities (44). How do Mn- and FeLOX adjust redox potentials to support lipoxygenation and β-fragmentation?

The first coordinating spheres of Mn- and FeLOX appear to be conserved (Table 3). The ionic radii of the oxidized metal ions are almost identical (45). In contrast to iron, manganese is always in high spin state in biological systems, and there is a strong Jahn-Teller distortion of the octahedral coordination during oxidation to Mn³⁺ (46). The second coordinating sphere and coordinated solvent molecules might play an important role in tuning the redox potentials (47, 48). This has been observed with superoxide dismutases of Escherichia coli with catalytic iron or manganese. Hydrogen bonds and steric factors adjust the redox potentials for Mn- and Fe- superoxide dismutases to similar values (45). The redox potentials of Fe- and MnLOX may be tuned by related mechanisms, but to resolve these issues will be a challenging task and was beyond the scope of this investigation.

Zonadhesin is known as a predicted protein with characteristic repeats of Thr, Ser, Val, and Ala residues at the C-terminal domain in analogy with adhesion molecules of Candida albicans and Saccharomyces cerevisiae (33, 35). Amino acid sequences with homology to zonadhesin_20-339 occur in peptidases and few other proteins of P. pastoris.⁶

Adhesion molecules of bacteria and fungi are crucial for cell-cell interaction, adherence to surrounding tissues, and the virulence of pathogens (34). Zonadhesin and several other fungal adhesins contain a ligand binding N-terminal domain of ∼300 residues and a C-terminal domain with repeats of Thr, Ser, and hydrophobic residues (34, 35). Zonadhesin_21-339 did not interact near the substrate channel of Gg-MnLOX. This may explain why we did not observe any apparent catalytic difference between preparations of recombinant Gg-MnLOX with and without zonadhesin present.

Bacterial adhesins, which are formed by Gram-positive bacteria, have been crystallized and the folds contain β-strands in analogy with IgG molecules [see (49) for review]. We used the Dali server (http://ekhidna.biocenter.helsinki.fi) to find similar structures to zonadhesin in the PDB and bacterial collagen adhesins yielded high scores with CNA of Staphylococcus aureus as the top candidate (50). Both zonadhesin and CNA contain two subdomains (N₁ and N₂; Fig. 11A). Each domain can be superimposed separately (Fig. 11B, C), but the orientations of the N₁ and N₂ domains differ in the two proteins.

Fig. 11.

Overview of the fold of zonadhesin_20-339 and the collagen adhesion protein, CNA, of S. aureus. A: Cartoon representation of the N₁ and N₂ domains of zonadhesin_20-339 in rainbow colors (N-terminal blue, C-terminal red; PDB entry: 5FX8). B: Cartoon representation after superpositioning of the N₁ domains of zonadhesin (rainbow colors) and CNA (gray color; PDB entry: 2F6A). C: Cartoon representation after superpositioning of the N₂ domains of zonadhesin (rainbow colors) and CNA (gray color). The two adhesins can be aligned with amino acid sequence identities of 13%, but the folds are similar in each domain.

We conclude that we have accidentally crystallized the first fungal adhesion protein with structural similarities to bacterial adhesins. The physiological function of zonadhesin is unknown, but it may adhere to proteins other than Gg-MnLOX by salt bridges and hydrogen bonds.

CONCLUSIONS

We report the crystal structure of Gg-MnLOX in complex with an adhesion protein (zonadhesin) at a resolution of 2.6 Å. The structure confirms that the metal ligands of Gg- and Mo-MnLOX are conserved, but the configuration and side pockets of the U-shaped active sites differ. The secondary coordinating sphere and hydrogen bonds likely tune the Mn²⁺ and Fe²⁺ metal centers to support catalysis. Zonadhesin appears to be the first crystallized fungal adhesin with structural similarities to collagen adhesins of Gram-positive bacterial pathogens.