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Originally published In Press as doi:10.1074/jbc.M800340200 on March 18, 2008

J. Biol. Chem., Vol. 283, Issue 20, 13897-13904, May 16, 2008
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An Oxidized Tryptophan Facilitates Copper Binding in Methylococcus capsulatus-secreted Protein MopE*Formula

Ronny Helland{ddagger}, Anne Fjellbirkeland§, Odd Andre Karlsen§, Thomas Ve§, Johan R. Lillehaug§, and Harald B. Jensen§1

From the {ddagger}Norwegian Structural Biology Centre, Faculty of Science, University of Tromso, N-9073 Tromso, Norway and the §Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway

Received for publication, January 14, 2008 , and in revised form, March 4, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35Å of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Copper is an essential nutrient for all living organisms, and molecular systems have been developed by all cells to maintain adequate supplies of copper. Copper-containing proteins play a key role in cellular respiration and are involved in biological processes such as pigment formation, neurotransmitter synthesis, and antioxidant defense. Recently, they have received increasing attention due to their role in human pathogenesis, in particular in neurodegenerative diseases such as Alzheimer, Parkinson, and prion diseases (1).

In Methylococcus capsulatus (Bath) and other methane-oxidizing bacteria, copper is important for both regulation and catalytic activity of the particulate methane-monooxygenase (2). An equivalent of this enzyme is used by almost all methanotrophs to catalyze the oxidation of methane to methanol, the initial and obligate step for all carbon fixation and energy production in these bacteria. A subset of methanotrophs, including M. capsulatus, produces a second soluble monooxygenase. Soluble monooxygenase does not require copper for activity and is produced only when the level of copper in the growth medium is very low (3, 4). The particulate methane-monooxygenase is the most growth effective of the monooxygenases, and hence, copper is required at relatively high levels for methanotrophs to grow optimally (3). Copper is most likely actively accumulated from the growth medium (3), and thus, the copper-homeostatic activity of methanotrophs differs from that of other prokaryotes in which systems handling extracellular copper is mainly involved in detoxification and elimination (5).

Both methanobactin and the MopE protein have been postulated to have a role in copper uptake in M. capsulatus (6, 7). Methanobactin is a small, siderophore-like compound that binds a single copper ion with high affinity (7). When copper is present in the growth medium, methanobactin is mainly associated with the membranes, possibly in direct association with the particulate methane-monooxygenase, whereas at copper-limited growth conditions, methanobactin accumulates in the growth medium. The MopE protein was originally identified as one of five outer membrane-associated proteins, designated MopA-E, Mop being short for M. capsulatus outer membrane protein (8). Later it was discovered that an N-terminal-truncated version (MopE*) of the cell surface-associated protein (MopEc) was secreted in large amounts to the growth medium under copper-limited growth conditions (6, 9). MopEc is composed of 512 amino acids, whereas MopE* represents the 336 C-terminal amino acids of MopEc. A MopE* homologue, designated CorA, has been isolated from the membranes of the methanotroph Methylomicrobium album BG8 (10). The expression of both MopE and CorA is negatively regulated by copper, indicating an involvement in copper-homeostatic activities. Importantly, a copper binding motif could not be predicted with significance from the primary sequence of the two proteins (9, 10).

In the present study we determined the crystal structure of MopE* to 1.35 Å of resolution and demonstrate that the protein has a novel protein fold and contains a mononuclear copper site. Importantly, we present evidence that one of amino acids in the copper-binding site is kynurenine, a tryptophan derivate not previously described in protein metal-binding sites.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Growth Conditions and Purification of MopE*—MopE* was obtained from spent medium of M. capsulatus (Bath) strain NCIMB 11132 grown in continuous cultures in nitrate mineral salts medium containing no added copper, as described previously (6), and purified as described in the supplemental information.

Protease Treatment and Mass Spectrometry Analyses—MopE* was digested with Lys-C endoproteinase (Roche Applied Science) (11, 12). Mass spectrometric analyses (MALDI-TOF2 MS and MS/MS) were performed at PROBE, University of Bergen. A detailed description is given in the supplemental information.

Metal Determination—The amount of copper bound per MopE* molecule was determined by inductively coupled plasma mass spectrometry (ICP-MS) at the Center for Element and Isotope Analyses, University of Bergen, Norway (see the supplemental information).

The Copper Binding Affinity of MopE—Information on the dissociation constant of copper binding to MopE* was obtained using the Cu(I) chelator bathocuproine. Bathocuproine disulfonic acid (Sigma) and ascorbate were added to MopE* (~10 µM) in 20 mM Tris-HCl (pH 7.5) and 1 mM CaCl2 to final concentrations of 0.5 and 1 mM, respectively. Ascorbate was included in the assay to ensure that the copper ions were maintained in the Cu(I) state. Samples were incubated either at room temperature or at 45 °C for 1 h with gentle shaking every 5 min. The protein was subsequently isolated using a 5-ml Hitrap desalting column (GE Healthcare), and the concentration of copper in the protein fraction was determined by ICP-MS analyses.

Cloning, Expression, and Purification of MopE* in Escherichia coli—MopE* was amplified by PCR and cloned into the pETM41 vector using BamHI and NcoI restriction sites. Large-scale protein expression was performed using E. coli BL21 StarTM (DE3) and the pETM41 vector. Purification of MopE* is described in the supplemental information.

Crystallization, Data Collection, Structure Determination, and Refinement—All variants of MopE* were crystallized from ammonium sulfate in the pH range 7.0–7.75. Data were collected at 100–120 K, and the data were processed in XDS (14), MOSFLM (13), and SCALA and TRUNCATE of the CCP4 suite (15). The structure was phased by single anomalous dispersion techniques using SHELXD (16) on a mercury derivative. The phases were improved by SHARP (17) and solvent flattening using SOLOMON (18). ARP/wARP (19) was used for automatic tracing of the protein, and the model was further improved by refinement using Refmac5 (21) and manual refitting of the model using O (20). The structures of MopE* and recombinantly expressed in E. coli (Rec-MopE*) were solved by molecular replacement using MOLREP (22). A more detailed description of the MopE* structure determination is presented in the supplemental information, and data collection and refinement statistics are summarized in Table 1.


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  		TABLE 1
Data collection and refinement statistics Values for outer shell are indicated in parenthesis

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The MopE* Structure—Crystals suitable for x-ray diffraction studies, with different morphology and cell parameters belonging to monoclinic (C2) and orthorhombic (I222) space groups, were obtained (Table 1 and supplemental Fig. S1). The crystal structure of MopE* was determined using single anomalous dispersion data to 1.9 Å collected on a HgCl2 derivative (Hg-MopE*). The structures of the wild-type protein (MopE*) and MopE* recombinantly expressed in E. coli (Rec-MopE*) were determined to 1.35 and 1.65 Å, respectively, by molecular replacement using the mercury derivative as starting model. Data collection and refinement statistics are provided in Table 1. The three structures of MopE* are essentially similar but with a significant difference in the metal-binding site.

The present structure (Fig. 1) includes the 290 C-terminal residues of the protein, and the structures have been refined to crystallographic R-factors of about 18–21% (Rwork) and Rfree of 20–24% (Table 1). Main chain atoms of MopE* and Rec-MopE* residues 47–336 superimpose on each other with a root mean square deviation value of 0.57 Å. The differences are caused by the loops formed by residues 75–80 (β2–β3 loop), 105–110 (β3–β4 loop), 115–119 (β4–β5 loop), and 308–312 (β19–β21 loop). Different crystal packing environments in the β2–β3 and β4–β5 loops and poorly defined electron density in the solvent-exposed β19–β20 loops of both proteins are expected to cause the different conformations in three of the four loops. Only the conformation of the β3–β4 loops is expected to be caused by differences in the metal-binding sites of the wild-type and recombinant proteins. The 46 N-terminal amino acids of MopE* were not identified in the electron density maps in any of the structures.

The protein forms a nest-like structure with dimensions of about 40 x 45 x 55 Å3 (Fig. 1, A and C). The majority of the MopE* structure folds into a coiled structure where only a third of the residues forms 21 β-strands (Fig. 1B). Eight of these build the only extensive secondary structure motif, an antiparallel β-sandwich. The MopE* structure also contains four 310 helical segments. The polypeptide is folded such that regions of the N-terminal, the middle, and C-terminal parts all are included in the β-sandwich (Fig. 1). One of the strands of the sandwich extends into a second 3-stranded sheet such that the central core of the protein is a 6-stranded sheet. Of the remaining β-strands, seven consist of only two residues each being involved in stabilizing the MopE* structure. The structure is also stabilized by a calcium ion octahedrally coordinated by three aspartic acid residues (Asp-250, Asp-252, Asp-274), two main chain carbonyl oxygens (Thr-276, Ala-279), and a solvent molecule (Fig. 1).


Figure 1
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  		FIGURE 1.
A, ribbon diagram illustrating the fold of the MopE* structure. The β-strands forming the secondary structure elements of MopE* are labeled B1 through B21, and the coloring follows the topology diagram described below. Copper is represented as a yellow sphere located between His-132, His-203, kynurenine 130, and a water molecule (red sphere) in a tetrahedral arrangement. A single calcium ion (green sphere) is coordinated by the side chains of Asp-250, -252, and -274 and the main chain carbonyl oxygen of Thr-276 and Ala-279. B, topology diagram (generated at the Topology of Protein Structure (TOPS) server, University of Leeds) illustrating the organization of secondary structure elements of MopE*. Triangles represent β-strands, and circles represent 310-helical segments. Blue triangles represent the β-sandwich, and cyan triangles represent the additional strands forming the central core. Red triangles represent strands consisting of only two amino acids. C, transparent van der Waals surface, including the ribbon representation, illustrating the crystal structure of MopE*. The ribbon is rainbow-colored, where blue is at the N terminus of the structure, and red is at the C terminus. Copper is represented as a yellow sphere.

 


Figure 2
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  		FIGURE 2.
Electron density maps of the copper-binding site of MopE*. The copper and the coordinating water, or hydroxyl, are illustrated as yellow and red spheres, respectively. The 2fofc electron density is contoured at 1{sigma}.

 
The copper-binding site contains a single partially buried copper ion (Fig. 1C) located in a trigonal arrangement by the side groups of His-132, His-203, and intriguingly, Trp-130. Tryptophan has been shown to bind various metals in chemical systems (23, 24); the indole side chain has not been reported to coordinate metal ions in biological systems. The electron density maps revealed a broken bond between the CD1 and NE1 atoms of Trp-130 (Fig. 2). In addition, the planar arrangement around the CG atom and the distances from the atom at the CD1 position to the main chain amine nitrogen atoms of residues 130, 131, and 132 suggested oxidation of the tryptophan to kynurenine. Kynurenines are formed by oxidative cleavage of the tryptophan indole ring with subsequent hydrolysis of the CD1 carbon, resulting in a mass increase of 4 Da compared with the unmodified tryptophan (Fig. 3B and supplemental Fig. S2).

The copper ion is also coordinated by an axial water molecule, forming a distorted tetrahedral arrangement of the copper-binding site with the copper ion located almost in the middle of the base of the pyramid and the solvent molecule in the apical position (Fig. 2). Trigonal, tetrahedral, and trigonal bipyramidal are all geometries previously observed for copper-binding proteins (25). The distances between the copper ion and the ND1 atoms of the histidines are in the range 1.99–2.17 Å, indicating covalent/ionic interactions. This is also in the same order as copper-histidine distances found in most other copper-binding proteins (26, 27). The distance between the copper ion and the nitrogen atom of the kynurenine is about 3 Å, somewhat longer than what is usually considered a copper-nitrogen interaction. The amino group of the kynurenine side chain would generally be considered a poor cooper ligand. The amino group is ~6 degrees off a linear phenyl-amino-copper interaction and, as such, does not coincide with a possible amino-metal interaction previously described (25). The copper to solvent distance in MopE* refines to about 2.5 Å, similar to what is found in other copper proteins. The occupancy of the copper ion is refined to about 0.65, which corresponds well with the ICP-MS analyses on purified MopE* revealing a copper-to-protein ratio between 0.5 and 0.6.

Identification of Kynurenine by Mass Spectrometry—To verify the oxidation of tryptophan to kynurenine, purified MopE* was digested with Lys-C, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. Computer-generated Lys-C peptide maps of MopE* calculated theoretical m/z ions of 2785 and 2789 for tryptophan and kynurenine, respectively. The MS analyses revealed a MALDI molecular ion at m/z 2789, consistent with the theoretically predicted ion for the kynurenine-containing peptide (Fig. 3, A and B, upper panels). Subsequent fragmentation of the m/z 2789 ion produced an MS/MS spectrum corresponding to the amino acid sequence Trp-112—Lys-135 of MopE* (Fig. 3C, left panel). From both the y-ion and b-ion series it was possible to identify a mass increase of 4 Da at residue 130. No distinct peak was observed at m/z 2785 (Fig. 3, A and B, upper panels).

Analyses of Recombinant MopE*—Crystallization and MS studies were also carried out on MopE* heterologously expressed and purified from E. coli (Rec-MopE*). The MS spectrum and MS/MS fragmentation pattern on peptides derived from Rec-MopE* revealed solely the unmodified m/z 2785 peptide, demonstrating that the post-translational formation of kynurenine in MopE* had not occurred when expressed in E. coli (Fig. 3, A and B, lower panels, and C, right panel). This was confirmed by crystal structure analysis of Rec-MopE*. The electron density clearly displayed an intact CD1–NE1 bond (Fig. 4A). In addition, the side chain was rotated about 60 degrees in {chi}1 and 180 degrees in {chi}2 relative to kynurenine in wild-type MopE* (Fig. 4B). To accommodate the tryptophan side chain in this position, the β3–β4 loop (residues 105–110) is displaced about 1.5 Å. Thus, the metal-binding site is sufficiently disrupted to prevent binding of copper in Rec-MopE*, as demonstrated by the lack of electron density at the wild-type copper position. The latter was confirmed by ICP-MS; Rec-MopE* did not bind detectable levels of copper. These findings substantiate that the conversion of tryptophan to kynurenine is an endogenous modification that specifically takes place in M. capsulatus and that the oxidation of Trp-130 to kynurenine is a prerequisite for copper binding in wild-type MopE*.

The Copper Binding Affinity of MopE*—Bathocuproine disulfonic acid was used to obtain information on the copper binding affinity of MopE*. Bathocuproine disulfonic acid forms a stable 2:1 complex with Cu(I) with an association constant of ~1020 (28). No significant decrease in the amount of copper bound to MopE* could be observed after treatment with the copper chelator either at room temperature or at 45 °C, thus indicating a high affinity binding (Kd < 10-20 M).


Figure 3
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  		FIGURE 3.
MALDI-MS spectra. A, MALDI-MS spectra of Lys-C produced peptides from MopE* (upper) and Rec-MopE* (lower) isolated from M. capsulatus and E. coli, respectively. Monoisotopic peaks are labeled with their respective m/z ratio. B, MALDI spectra (mass range 2783–2796 Da) of A) showing the m/z 2789 (upper) and m/z 2785 (lower) ions. Right-hand side, chemical structures of kynurenine and tryptophan. C, tandem mass spectra of the m/z 2789 ion (left panel) and the m/z 2785 ion (right panel), indicating the observed fragmentation pattern and the sequence ion assignments. Predicted ions are shown at top of the figures, where a 4-Da increase in mass at Trp-130 is considered for the m/z 2789 ion. The oxidized tryptophan is displayed as kyn in the amino acid sequence of the 2789-Da peptide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We present here x-ray diffraction and mass spectrometry data on the M. capsulatus-secreted protein, MopE*. The protein presents a unique kynurenine-containing copper-binding site and a novel protein fold, of which only about a third of the MopE* structure displays similarity to known proteins. The only extensive secondary structure element previously observed is an 8-stranded antiparallel β-sandwich formed by about 25% of the residues. This motif is often found in virus capsid proteins and sugar binding and hydrolyzing proteins as well as in oxidoreductases, cupredoxins (proteins with Type I copper center), and proteins involved in electron transport (DALI data base (29), ProFunc (30), and CATH protein classification data base (31)). The remaining residues form an extensive coil-like structure interspersed with 13 short β-strands. Particularly interesting is the copper-binding site located in a depression on the molecular surface about 8–10 Å from the β-sandwich and relatively distant from any secondary structure element. The partially buried copper ion is located between two histidines and an oxidized tryptophan (kynurenine) in a planar trigonal arrangement and displays, therefore, similarities to Type I (blue copper proteins) and CuB copper centers (25, 32). Type I copper centers are characterized by two histidines and a cysteine in a planar trigonal arrangement and a variable axial ligand. CuB copper centers coordinate copper by three histidines in a trigonal pyramidal geometry, and the axial ligand forms a bridge to the iron ion of the cytochrome heme. Two and three histidine ligands are frequently observed in copper-binding proteins, but to our knowledge this is the first observation of a tryptophan or a tryptophan metabolite involved in metal binding.

When previously detected in proteins, kynurenine was considered the result of oxidative damage, and in most studies on proteins containing modified tryptophan residues, the kynurenine modification was formed by exposing the proteins to oxidative stress in vitro (33–35). There is accumulating evidence that oxidation of specific tryptophan residues takes place in vivo (36–40), but evidence for biological functions of kynurenines in proteins has to our knowledge not been presented. Structural evidence by NMR for naturally occurring kynurenine has been provided only for the antibiotic peptide daptomycin (37, 38) from Streptomyces roseosporus. In other studies on kynurenine-containing proteins, kynurenine was detected by MS analyses, and for all proteins examined the MS spectra revealed two distinct peaks with m/z corresponding to oligopeptides containing both unmodified and modified tryptophan residues (35, 36, 39, 40). We did not detect the tryptophan signature peak at m/z 2785 in the mass spectrum of MopE* (Fig. 3, A and B) in our present study. This indicates that oxidation of the specific tryptophan to kynurenine in MopE* is highly efficient in M. capsulatus. The mass increase of 4 Da observed for the modified tryptophan is compatible with an opening of the indole ring resulting in an amino group attached to the phenyl ring and a methyl group at the aliphatic chain (supplemental Fig. S2C), but this would not be consistent with the planar arrangement observed around the atom corresponding to the CG position of a Trp-130. A methylene group would fit the planar arrangement (supplemental Fig. S2D), but this molecule would present a mass increase of 2 Da relative to tryptophan. The relatively short distances from the atom at the possible methyl group corresponding to the Trp-130 CD1 position and the main chain nitrogen atoms of residues 131–133 (2.9–3.2 Å) correspond more to hydrogen bond distances than a polar-nonpolar N-C interaction. Thus, our x-ray diffraction data are consistent with the presence of kynurenine also in the MopE* crystals.


Figure 4
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  		FIGURE 4.
A, electron density maps covering the residues forming the copper-binding site of Rec-MopE*. The 2fofc electron density is contoured at 1{sigma} and shows that Trp-130 is in its unoxidized state. B, MopE* (yellow) superimposed on Rec-MopE* (green). Trp-130 in Rec-MopE* is rotated relative to Kyn130 in MopE. The copper found in MopE* is illustrated as a yellow sphere.

 
Metal binding distances are generally considered a function of the size, ionization, and electron donating properties of the ligand. The distance between the copper ion and the nitrogen atom of the kynurenine is about 3 Å, considerably longer than what is usually considered a copper-nitrogen interaction, which is 2–2.3 Å (see i.e. (26, 41)), and longer than some of the longest Cu–N bonds reported previously (in non-biological systems; 2.8 Å (42)). The kynurenine is, therefore, probably not a first-sphere ligand, but the ring amino group is close enough to be a second sphere copper ligand.

The copper content of purified MopE* and crystallized MopE* were similar (0.5–0.6 and 0.65 copper per molecule MopE*, respectively), which was less than what would be expected given the high copper affinity determined for MopE*. Purified MopE* was unable to bind additional copper, added either as Cu+ or Cu2+ (data not shown). The actual copper content of purified MopE* was found by ICP-MS analysis to depend largely on the copper content of the growth medium but did not exceed about 80% of the sites.

In contrast, when co-crystallizing MopE* (Cu-MopE*) with 10 mM CuSO4, the occupancy of copper increased from ~65% to 100%. Under copper-containing conditions crystals belonging to a new monoclinic space group (C2; 104.11 x 101.58 x 38.64 Å3, β = 101.40°) have been obtained. These crystals diffracted to 1.6 Å (data not shown). The addition of copper to the crystallization conditions had only minor effects on the binding distances, with differences to the neighboring amino acids less than 0.1 Å. The copper-binding site is open to the solvent in both crystal forms and should in principle not be significantly influenced by close crystal packing interactions. The differences are in the same order as the coordinate error (0.06 Å–0.08 Å), and hence, no trend related to copper occupancy could be drawn.

A possible explanation for the difference in metal binding capacity between purified (65%) and crystallized MopE* (65–100%) could be related to structural differences and may to some degree reflect the amount of copper bound during expression and retained through purification. In the crystal, the 46 N-terminal amino acids are invisible in the electron density maps in all crystal forms, suggesting that this domain is unstructured or cleaved off during the crystallization process. We hypothesize that in solution the N-terminal domain of MopE* may serve as a lid regulating/inhibiting further metal binding. Removal of this domain would expose the binding site to the solvent, allowing copper binding in all sites.

The kynurenine amino group is ~6 degrees off a linear phenyl-amino-copper interaction and as such does not fit the amino-metal interaction requirements described by Holm et al. (25). The kynurenine residue is, therefore, apparently not a ligand in the classical sense, and at present we can only speculate about the function of kynurenine in the copper-binding site of MopE*. Because the Trp-130 of MopE* expressed in E. coli is not oxidized and this protein does not bind copper either in solution or in crystals grown in the presence of copper, it is likely that the conformational changes that take place as a consequence of Trp-130 oxidation to kynurenine is a requisite for copper binding. The solvent-exposed indole side chain of Trp-130 in Rec-MopE* is stabilized only by a hydrogen bond to Asp-105 and by weak hydrophobic interactions to neighboring residues. After oxidation, rotation of the kynurenine side chain allows stabilization of the carbonyl oxygen through hydrogen bonds to the main chain amine nitrogen atoms of residues 131–133. Kynurenine in this conformation reduces the size and flexibility of the otherwise relatively open copper-binding site, possibly providing a structural pocket for entrapment of the copper ion and, thus, ensuring efficient covalent/ionic binding to the histidine ligands.

The chemical properties of Trp are considered incompatible with direct metal-coordination in biological systems (24). Consistent with this, the E. coli expressed Rec-MopE* containing only unmodified Trp-130 did not bind detectable levels of copper. The lack of Trp modification in Rec-MopE* is intriguing because it suggests that this modification is enzymatically mediated and consequently most likely requires another protein(s) to catalyze the reaction. In this respect, it is interesting that the mopE gene (MCA2589) is located in an operon with the MCA2590 gene encoding a protein displaying sequence similarity to the tryptophan-modifying MauG proteins (6, 43). The MauG proteins are c-type cytochromes that are required for biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase (44, 45). The M. capsulatus MauG homologue is, like MopE*, located on the surface of the bacterium and is a member of a novel group of the bacterial di-heme cytochrome c peroxidase family with unknown function (43). The MCA2590-encoded cytochrome may be active in the conversion of Trp-130 to kynurenine, but this possibility remains to be investigated. Interestingly, a protein with 50% sequence similarity to the M. capsulatus MCA2590-encoded cytochrome is predicted from an unannotated ORF located immediately downstream of CorA, the MopE* homologue produced by the methanotroph Methylomicrobium album BG8 ((10) see below).

The structure of MopE* displays little resemblance to known copper proteins, and the function can, therefore, not be predicted from the structure. To date, the copper-repressible CorA protein is the only known MopE homologue. CorA is composed of 204 amino acids, all of which can be aligned to the C-terminal region (amino acids 56–336) of MopE* (9) and with conserved residues around the metal-binding sites (supplemental Fig. S3). Also, the three aspartic acid residues that contribute side chain coordinates for the calcium ion in MopE* (Fig. 1, supplemental Fig. S3) are conserved in CorA. Thus, circumstantial evidence indicates that MopE and CorA have a related function in copper homeostatic activities. Because MopE is copper-repressed and has a very high affinity for copper, it is tempting to speculate that MopE plays a role in copper uptake and transport at low copper culture conditions. Very little information about copper uptake in methanotrophs is available. The only molecule in addition to MopE that has been linked to copper transport in these bacteria is methanobactin (7). Like MopE*, methanobactin is accumulated in the medium at low copper growth conditions and binds a single copper with high affinity, similarly to MopE*. A low Kd would be expected for a copper-binding protein operating in a low copper environment. If MopE is involved in copper transport, a copper receptor is likely to exist at the cellular surface, but candidate molecules mediating intracellular transport remain to be identified, and characterization of MopE participation in copper homeostasis must await further studies.

The finding of kynurenine as oxidized tryptophan residues in biological systems has so far been described from studies related to oxidative stress or exposure to reactive oxygen species (33–40). In the case of E. coli-expressed MopE*, Trp-130 was not oxidized to kynurenine when exposed to strong oxidizing conditions. This could indicate that the oxidation of Trp-130 in M. capsulatus is enzymatically catalyzed. If so, it is tempting to speculate that MopE* is involved in a redox process important for the uptake of copper ions, which most likely takes place at the surface of the bacterium. Involvement in a transport process would explain the reason for choosing a weak ligand, such as kynurenine, because easier transfer of the copper ion to its receptor molecule would be anticipated. The receptor molecule would still have to induce chemical and/or conformational changes to MopE* to release the copper ion. Experiments are initiated to elucidate these questions and unravel whether the role of kynurenine in copper binding is a unique trait of MopE restricted to proteins of methane-oxidizing bacteria or whether it is a more frequent structural feature in biological metal coordination.

The identification of the kynurenine reported here is an important observation of a structural difference between a wild-type protein and its heterologously expressed counterpart. It, thus, provides a warning example that a heterologously expressed protein does not always exert the same properties as that from the wild-type organism. In the case of MopE, the modification takes place only when endogenously expressed in M. capsulatus and, thus, appears to be linked to its biological function. In this case the general use of recombinant proteins for structure studies may have made the detection of the kynurenine elusive.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 2VOV, 2VOW, and 2VOX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The present study was supported by the national Functional Genomics Program (FUGE) and other grants from The Research Council of Norway. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 To whom correspondence should be addressed: Dept. of Molecular Biology, HIB, University of Bergen, Thormøhlensgate 55, N-5020 Bergen, Norway. Tel.: 47-55-58-64-22; Fax: 47-55-58-96-83; E-mail: harald.jensen{at}mbi.uib.no.

2 The abbreviations used are: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectroscopy; ICP, inductively coupled plasma. Back


   ACKNOWLEDGMENTS
 
Provision of beamtime at the MX beamlines at the European Synchrotron Radiation Facilities (ESRF), the Swiss-Norwegian Beamlines at ESRF, Swiss Light Source and BESSY, and the access to the Centre for Element and Isotope Analyses facility at the University of Bergen is gratefully acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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