Introduction

Multicopper oxidases (MCOs) are a large, widely distributed and diverse family of enzymes with various functions ranging from copper (Cu) and iron metabolism to polyphenol oxidation1. One important feature of MCOs is that a minimum of four Cu ions are arranged at two sites: the mononuclear type 1 Cu centre (T1), and the trinuclear Cu cluster (TNC) consisting of a type 2 Cu centre (T2) and a dinuclear type 3 centre (T3)1,2. Four single electron-transfer reactions from the T1 site to the TNC cluster are coupled to the oxidation of various substrates. Electrons are passed to dioxygen bound to the TNC to generate two water molecules.

Laccases are the largest subfamily of MCOs and widely distributed in diverse organisms. They have multiple functions, including lignin biosynthesis and wound healing in plants; lignin degradation, pigmentation, and pathogenesis in fungi; and pigment production in the endospore coat in some bacteria such as Bacillus subtilis3. Laccases can catalyse the oxidation of a broad spectrum of organic substrates such as polyphenols, diamines, and some inorganic compounds4. These enzymes have been received increasing attention because of their practical applications in the textile, food, and wood-processing industries and their possible uses in bioremediation5.

CueO, an MCOs from Escherichia coli (E. coli), together with CopA, a P-type ATPase, comprises a copper efflux (cue) system that resists copper stress under aerobic conditions6,7. Expression of both enzymes is stimulated by exogenous Cu ions via the cytosolic metalloregulatory protein CueR8,9. Previous analysis of the crystal structure of E. coli CueO has highlighted that it adopts a canonical architecture consisting of three similar cupredoxin-like domains linked by peptides. This architecture is shared by the other known laccase and ascorbate oxidase. Within domain III, a unique 42-residue methionine-rich region made up mainly of methionine-rich helix (thereafter, MR helix) may hinder the access of bulky organic substrates to the T1 Cu site10. This region near the T1 Cu position was later found to coordinate a labile fifth Cu atom, a novel feature of CueOs that differs from other MCOs11. Like other common laccases, CueO exhibits phenol oxidase activity with a broad range of substrates. However, this enzyme possesses an unique cuprous oxidase activity in vitro6,12. CueO can catalyze the oxidation of cuprous ion (Cu(I)) to the less toxic cupric ion (Cu(II)) in vivo12.

Recent structural and functional studies of engineered E. coli CueO have showed that removal of the methionine-rich helical region significantly decreased cuprous oxidase activity of the enzyme, but increased its phenol oxidase activity. These findings implied that the MR helix region confers the specific cuprous oxidase of CueO13. Djoko et al. provided compelling evidence that the higher affinity of the methionine-rich region for Cu (I) over Cu (II) explains why CueO functions solely as a cuprous oxidase but not phenol oxidase in vivo5B). Although the precise passageway through hole to T1 Cu site has not characterized, it is predicted that G304K will show markedly higher laccase activities for organic substrates, compared with that of the wild-type. Noticeably, in G304K in the absence of Cu ions, the hole formed by the hydrophobic pocket without fully trap** the R-loop was still open and allow the passage of organic substrates (Fig. S4A,B).

Figure 5
figure 5

Structural comparison among different versions of CueO, including E. coli CueO (PDB entry 1N68) (A) G304K mutant (B) and Δα5–7 (PDB entry 2YXW) (C). The structures are represented in cartoon (left) and electrostatic surface (right). Domain I-III is colored green, palecyan, and magenta, respectively. In electrostatic surface, red present negative electrostatic potential; blue positive electrostatic potential. For the electrostatic surface presentation of G304K mutant, Cu5 and Cu6 are shown in copper spheres.

Full size image

Catalytic properties of mutants derived from G304K

In most of the reported CueO structures10,11, the loop following the MR helix has no electron density. This implied that the region is disordered and that it is the most flexible part of CueO structure. Only in the complete structure of CueO at a resolution of 1.1 Å (PDB entry 3OD3) is the previously unseen loop fully resolved15. Comparative analysis of the G304K structure with the complete CueO structure indicated that the resolved loop following the MR helix in the complete CueO structure is in close proximity to the dramatically wiggled R-loop in the CueO mutant structure (Fig. 6A). In the light of that finding, together with the above structural insights, we hypothesized that the hydrogen bonding and hydrophobic force may mediate the specific interaction between the R-loop and the disordered loop following the MR helix in G304K structure, resulting in the dramatic movement of the MR helix.

Figure 6
figure 6

Analysis of laccase activity in six double mutants derived from CueO G304K mutant evidences that the R-loop modulates the enzyme activity. (A) G304K mutant structure superimposed on complete structure of wild-type protein (PDB entry 3OD3). Top shows superimposed overall structures; Bottom highlights relationships among R-loop, MR helix and disordered loop in G304K and wild-type enzyme. Residues are shown as sticks. (B) Diagram of several motifs and mutation points in CueO proteins. SS indicates signal sequence. R-loop is regulatory loop. Mutated residues in different mutants are shown in red. MR helix refers to methionine-rich helix. (C) Maximal velocities showing laccase activity of mutants shown in (B) and wild-type enzyme. Maximal velocities were measured using ABTS as substrate at pH4.5 and 37 °C. Data are mean ± standard deviation from at least three experiments.

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On the basis of this rationale, we cloned and purified six double mutants (including M1-M6) based on G304K mutant and measured the laccase activities of these mutants (Fig. 6B). The steady-state laccase activities of the wild-type CueO and mutants were measured using ABTS as the substrate. The resulting Km and Vmax values are listed in Table 2. The G304K mutant enzyme showed much higher catalytic activity than that of the wild-type, consistent with our previous findings16 (Fig. 6C). M1, which contained consecutive four point mutations, had lower catalytic activity than that of G304K. Intriguingly, among the four double mutants M2-M5, M5 had lower activity than that of G304K, but similar activity to that of the wild-type. This suggested that Gln383 plays a critical role in enhancing the interaction between the R-loop and the disorder region possibly by making hydrogen bond with Lys304 (Fig. 6C). Unexpectedly, M6 showed substantially higher laccase activity than those of the wild-type and G304K (Fig. 6C). It is speculated that in M6, not only the hydrogen force between Lys304 and Gln383, but also the hydrophobic interaction between consecutive mutated alanine residues and the hydrophobic residues on the wiggled R-loop, such as Met303, Met305, and Ile307, may reinforce the swing of MR helix. Therefore, in M6, the hole around the access to T1 Cu site is expected to become wider to accommodate bulkier organic substrates, to enhance the catalytic activity.

Table 2 Kinetic parameters of the wild-type CueO and various CueO mutants for ABTS oxidase activity.
Full size table

To observe the effects of Cu-induced conformational changes in the G304K structure, we added Cu (II) ions and then determined the laccase activities of G304K and important mutants derived from it, including M5 and M6. The addition of Cu led to great increases in the laccase activity for G304K than for the wild-type enzyme, implying that the mutant had better stronger Cu-binding ability than that of the wild-type CueO (Fig. S6). The assay results were consistent with the results of structure analysis, that is, that the addition of Cu makes the hole (which is formed by the hydrophobic pocket without trap** the R-loop) large enough to allow the passage of bulky organic substrates. The addition of Cu similarly increased the laccase activity of M5, M6 and G304K (Fig. S6).

The wild-type CueO is an excellent cuprous oxidase12. We conducted the cuprous oxidase activity assay for the wild-type CueO and important mutants derived from it including CueO G304K, M5 and M6. The cuprous oxidase activity of G304K mutant (Vmax = 6.3 U/mg) was about 20% that of the wild-type CueO (Vmax = 29.5 U/mg), while the Km value for G304K mutant (Km = 80.3 uM) was four times lower than that of the wild-type CueO (Km = 427.8 uM) (Fig. S7). The kinetic parameters determined for the cuprous oxidase activity of M5 and M6 were similar to those of G304K (Fig. S7). Thus, the cuprous oxidase activities of these important CueO mutants were also inhibited.

Discussion

Methionine-rich regions are found in the sequence of numerous proteins involved in Cu homeostasis, such as CueO10, PcoA28. The methionine-rich region in the CueO lies over the T1 Cu site, so it can interfere with the access of substrate to T1 Cu site10. This region harbours multi-Cu-binding sites to confer its cuprous oxidase activity29. The loss of the complete methionine-rich region do not change the overall structure, but significantly affects the enzymatic activity13,14, especially laccase activity, suggesting that the methionine-rich region is very important for activity. However, how the enzyme activity is regulated via methionine-rich region is not fully understood.

In our previous work, we isolated and characterized the E. coli CueO mutants from the sludge in a chemical plant, among which G304K had 2.7-folds higher for activity than that of the wild-type16 (Fig. 6C). The finding is surprising, because in CueO structure, Gly304 located on the R-loop was 14 Å away from the T1 Cu site, and mutation adjoining the catalytic sites in the other point mutants such as M441L generally results in the loss of enzyme activity11.

To determine the reason for the change of enzyme activity, we analysed the crystal structure of G304K. Compared with the wild-type enzyme structure, G304K showed dramatic conformational changes in the MR helix and R-loop (Fig. 3). Interestingly, binding of Cu ions induces the local conformational changes, especially the sway of the R-loop (Fig. 2). Obviously, the sway of the R-loop triggered significant movements of the methionine-rich region, resulting in the appearance of a hole through which the substrate could access to T1 Cu site (Fig. 5B). The structural changes may explain the increased laccase activity of the mutant enzyme.

Functional analyses of CueO double mutant confirmed the interaction between the R-loop and the methionine-rich region. In addition, cuprous oxidase activity was inhibited in the CueO mutants G304K, M5 and M6. Although the coordinating residues for sCu were still intact in the CueO mutant structure, the coordinating environment for sCu had changed and no electron density which could be assigned as Cu was detected at the sCu-binding site. The conformational change resulted from the conversion of Gly residue into Lys residue on the R-loop, which may have affected the cuprous oxidase activity of the CueO mutant. Collectively, our results define how the R-loop regulates the enzyme activity by modulating the methionine-rich region.

In Cu-binding metaloenzymes, amino acid residues coordinating Cu atoms in the proteins include His, Met, Cys, Asp and Glu1,30; however, histidine ligation with Cu is by far the most frequent1,31. We and others11,13 have found some novel Cu-binding sites on the surface of the CueO crystal structure, apart from the essential catalytic Cu atoms buried in the core domains. In our research, only His was found to coordinate with Cu5 and Cu6, whereas His, Asp and Glu were found to coordinate with Cu7 and Cu8 (Figs 1 and S1A,B), as has been reported for the previously described structures of CueO11,13 (PDB entries 1N68 and 2YXW).

Our results raised the interesting question as to the role of Cu-binding sites on the surface of the wild-type CueO and G304K mutant enzyme. On the basis of the results of our study and previous studies, we suggest that Cu-binding sites on the surface of CueO have at least two practical roles: to allow for the transfer of Cu ions among Cu-binding proteins; and to induce a conformational change in the CueO structure. Previous studies have shown that CueO is localized in the periplasm of E. coli cell, along with the other Cu-binding proteins such as bacterial MCO PcoA28 and its putative partner PcoC32. Moreover, it is generally thought that the Cu atoms in the Cu-binding proteins are well poised for metal transfer because of their low coordination number and their solvent accessibility33. The coordination number Cu5, Cu6 and Cu8, which are bound to the surface of CueO mutant, was 3, whereas it was as low as 2 for Cu7 (Figs 1 and S1A,B). This suggests that Cu atoms may be easily transferred among the Cu-binding proteins, including CueO, PcoA, and PcoC, within the periplasm of bacteria cells. Further research is required to explore the implications and mechanism of this transfer. The second possible role of surface Cu-binding sites is to change the conformation of the CueO structure. The structural comparison of G304K with and without excess Cu ions showed that with the excess Cu ions, Cu5, Cu6 and the other Cu atoms occupied the sites on the surface, the R-loop displaed dramatic movements, and the imidazole group of His143 rotate 180° upon incorporation of Cu atom into Cu3 position (Fig. 2). To our knowledge, it is the first time that Cu atom have been shown to change CueO structure. Our analyses showed that the laccase activity of the CueO mutants was dependent on Cu, suggesting that it is mainly Cu5 occupancy that makes the hole large enough to allow the passage of bulky organic substrates to enhance laccase activity.

In summary, analyses of X-ray crystal structures of G304K revealed that the R-loop, without being trapped in the pocket, regulates the catalytic activity of CueO by giving rise to a local conformational change around the MR-helix. Extensive engineering of the protein based on the R-loop and the unstructured methionine-rich region are needed to generate substrate-specific CueO mutant for potential applications in biotechnology.

Methods

Cloning, Expression and Purification

Using the standard PCR cloning strategy, the truncated CueO G304K gene (amino acids 29–516) lacking the signal sequence-encoding region from the metagenome was cloned into the NdeI and XhoI sites of modified pET28a (+TEV) vector. The construct was confirmed by DNA sequencing.

The recombination CueO G304K gene was overexpressed as 6× His-fusion protein in Escherichia coli strain Rosetta2 (DE3) pLysS (Novagen). E. coli cultures harboring fusion protein were incubated in the shaker at 37 °C at 220 rpm. Subsequently, the shaker temperature was shifted to 25 °C, when its density reached an OD600nm of 0.8. The expression of recombination proteins was induced with the supplement of isopropylb-D-thiogalactoside (IPTG) 1.0 mM plus CuCl2 0.25 mM final concentration, respectively. After growth for further 1 h at 25 °C at 160 rpm, the cultures were kept at static incubation until harvest. The cell sediments were suspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 20 mM imidazole and 500 mM NaCl, and disrupted in a French press at 12,000 psi for three cycles. To remove cell debris, the lysate was centrifuged for 40 min at 13,000 × g. The supernatant were collected and loaded on the equilibrated Ni2+ Chelating SepharoseTM Fast Flow (GE Healthcare). After several gradually crush wash, CueO G304K protein was eluted with a buffer containing 20 mM Tris-HCl pH 8.0 and 500 mM NaCl supplemented with 300 mM imidazole. The eluate was digested using TEV protease to remove 6× His tag, while dialyzed against 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, overnight at 4 °C. The eluate was filtered using a 0.45 μm filter and concentrated prior to loading onto a 25 ml superdexTM 200 10/300 GL (GE Healthcare). The peak fraction was pooled and concentrated to ~15 mg/ml for crystallization.

The other CueO mutants were purified according to the same protocol as for CueO G304K protein. Purified CueO protein and derived mutants were analyzed by SDS-PAGE for purity.

Crystallization and X-ray Data Collection

CueO G304K protein was crystallized by the sitting-drop vapor diffusion method at 22 °C, with 0.7 μl 15 mg/ml protein solution mixed with an equal volume of reservoir solution. Initial crystallization trials screen needle-shaped crystals in a mother liquor containing 0.2 M potassium formate (pH 7.0), 20% (w/v) Polyethyleneglycerol 3,350. Further optimization of the crystallization condition led to the appearance of rod-shaped crystals in the well in which 0.7 μl 10 mg/ml protein solution was mixed with an equal volume of reservoir solution containing 0.2 M potassium formate (pH 8.5), 20% (w/v) Polyethyleneglycerol 3,350. Crystals were transferred to 30% Polyethyleneglycerol 3,350 prior to flash-frozen in liquid nitrogen. For Cu-protein complex, crystals were soaked in the identical solution plus 10 mM CuCl2 for 60 min, and transferred to 30% Polyethyleneglycerol 3,350, and flash-frozen in liquid nitrogen.

The data set of CueO G304K protein was collected at beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). The data set of CuCl2-soaked protein was collected at beamline BL1A of Photon Factory (PF) at the wavelength of 1.1 Å, and the anomalous data was collected at the Cu absorption edge of the wavelength of 1.37 Å at beamline BL5A of PF. The data were indexed, integrated and scaled with HKL200034.

Structure determination and refinement

The initial phases determined by molecular replacement using the program Phaser35 from CCP4 program suit36, with the crystal structure of full length E. coli CueO (PDB entry 1N68) as a searching model.

The structure refinement was carried out with Phenix37 and Refmac38. Model building was carried out by iterative rounds of manual building with COOT39. According to the anomalous difference Fourier maps, the locations of Cu ions were identified. MolProbity40 was used to validate the structure. Data collection and refinement statistics are listed in Table 1.

All structural figures were rendered using PyMOL programme (http://www.pymol.sourceforge.net/). For comparisons, the structures were superposed using the program LSQKAB of the CCP4 suite36.

Absorbed dose calculation

Absorbed dose calculations were performed using RADDOSE-3D25,41. The values of the beam parameters (including the energy, flux, profile, spot size, etc), crystal properties (unit cell, space group, number of molecules per asymmetric unit, composition, size and thickness of the crystal), and wedge parameters (angle of the rotation, exposure time, the angular step size) were provided as the input information. Among the values, the beam energy was 12.1KeV, the beam flux was 4.1 × 1012 photons per second, and exposure time was 36 second25.

The copper content and electron paramagnetic resonance spectrum

The concentration of protein in enzyme pools was routinely determined by the method of Bradford using bovine serum albumin as the standard. Copper content was determined by X-series ICP-MS (inductively coupled plasma mass spectrometry) (ThermoElemental, Beverly, Mass.) operated by the Key Laboratory for Biomedical Effects of Nanomaterial and Nanosafety of Institute of High Energy Physics from CAS. X-band electron paramagnetic resonance (EPR) spectra were recorded on a BrukerESP 300 spectrometer (Billerica, Mass.) operated by State Key Laboratory of Natural and Biomimetic Drugs of Peking University.

Mutagenesis of CueO

Site-directed mutagenesis of CueO was performed as previously described42. Briefly, pET30-CueO_G304K was used as a template. Desired mutations of CueO_G304K were amplified by complementary mutagenic primers (M1F/R-M6F/R) (S Table 2) with FastPfu DNA polymerase (TIANGEN, Bei**g, China). The PCR product was treated with Dpn I to remove the parental template DNA and introduced into E. coli DH5α. In this way, the pET-CueO_G304K derived plasmids pET-M1, pET-M2, pET-M3, pET-M4, pET-M5 and pET-M6 with specific mutations (S Table 1), verifying by DNA sequencing, were obtained. All these plasmids were transformed into E. coli BL21(DE3) for protein expression.

Laccase activity assay

The substrate ABTS [2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)] (sigma) was used to assess the laccase activity of the wildtype CueO and various CueO mutants by monitoring the change in absorption at 420 nm as described previously16. The assay mixture was consisted of 100 mM citrate/phosphate (pH 4.5) or 100 mM sodium acetate (pH 4.5), 2 mM ABTS, specified or varied concentration of CuCl2 at 37 °C. To initiate the reaction, purified enzymes were added at the final concentration 3 nM.

One unit of enzyme activity was defined as the amount of the laccase that oxidized 1 μmol of ABTS substrate per min. Activity measurements were plotted as a function of substrate using GraphPad 5.0 (La Jolla, CA) and kinetic constants, Km and Vmax, were evaluated using a leastsquares fit of a Michaelis-Menten curve.

Cuprous oxidase activity assay

The cuprous oxidase activities of the wild-type CueO and various CueO mutants were measured indirectly by monitoring oxygen consumption using a Clark-type oxygen sensor electrode (Hansatech, Cambridge, UK), with [Cu(I)(CH3CN)4]PF6 (Sigma) as a substrate, as described by Singh et al.12.

In brief, stock solutions of the substrate were freshly prepared in degassed and nitrogen-purged acetonitrile. To initiate the reaction, a 100 ul aliquot of Cu(I) substratewith varied concentration, was added to an air-saturated mixture containing 312 nM enzyme, 100 mM Tris-acetate (pH5.0), 5% acetonitrile, and 0.2 mM Cu(II). All kinetic measurements were made at 18 °C and pH 5.0. Enzyme velocities were calculated after subtracting the aerial oxidation of Cu(I) in the absence of enzyme. Activity measurements were plotted as a function of substrate using GraphPad 5.0 (La Jolla, CA) and kinetic constants, Km and Vmax, were evaluated using a least squares fit of a Michaelis-Menten curve.

Accession numbers

The atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://wwpdb.org/) with accession codes 5YS1 (CueO G304K mutant) and 5YS5 (Cu-soaked CueO G304K mutant).