Enzyme activity of CopA
As shown in Fig. 1, a purified band (lane 5) at approximately 60.0 kDa was observed by SDS-PAGE analysis. The molecular mass of CopA is 58.3 kDa calculated by software ExPASy (https://web.expasy.org/cgi-bin/compute_pi/pi_tool) and the vector pET-28a(+) has 21 amino acids about 2.3 kDa. Therefore, the fusion protein mass is about 60.6 kDa, which is consistent with the experimental results. By comparing lane 5 with lane 2 and lane 4, the target protein CopA was successfully enriched and purified by Ni Focurose 6FF column and DEAE Focurose 6FF column. The WB analysis results of the protein samples are shown in FIG S1, the band of lane3 is significantly larger than lane1, indicating that the protein concentration is greatly improved after purification, which is consistent with the results of the BCA test. The lane2 test was negative, and lane1 and lane3 were positive, indicating that the fusion protein CopA is immunologically active.
We put CopA purified from BL21-pET-copA which cell grew in two conditions without Cu(Ⅱ) and with 0.25 mM Cu(Ⅱ) into reaction systems which contain 1 mM Cu(Ⅱ). As shown in FIG. 2A, lots of brown precipitates generated in tube 2 and tube 4 and they all colored upon addition of LBB reagent (FIG 2B). Surprisingly, in addition to tube 2 and tube 4, tube 5 also turned blue, indicating that the BL21-pET-copA in copper-free culture condition produced CopA which still has manganese oxidation activity although weakened. This is inconsistent with the report Mnx (Butterfield and Tebo 2017). Without the addition of extra Cu(Ⅱ) to the mnx-expressing E. coli growth medium, Mnx will precipitate during dialysis and is inactive. The produced Mn oxide were quantified spectrophotometrically using Microplate reader (Saputra et al. 2013). The manganese oxide yield of tube 4 is 11.2 times that of tube 2 and 114 times that of tube 5. This shows that Cu(Ⅱ) has a great influence on the manganese oxidation ability of CopA.
Sequence analysis of CopA
The similarity of amino acid sequence is not high (less than 31%) between CopA and known manganese oxide protein (Zeng et al. 2018), which may lead to different oxidation mechanism. But MCOs almost all contain four highly conserved copper ion binding sites and a Cys and 10 His and its surrounding amino acid ligands linked to copper atoms are relatively conservative as shown in Fig. 3a, suggesting that they may have similar biological functions, that is, they all have manganese oxidizing ability. The copper-binding domain of the copper oxidase is usually in the form of HXH (H is histidine) as shown in Fig. 3a. In Fig. 3b, the A and B sites of CopA, are located near the N-terminus of the protein like other MCOs (MofA, MoxA, CotA, CueO), and the C and D sites are located at the C-terminus. However, the sequence feature of MnxG are reversed, with the A and B sites at the C-terminus and the C and D sites at the N-terminus (Dick et al. 2008).
Manganese removal efficiency of mutant strains
Figure 4 shows manganese removal efficiency of BL21-pET-copA and mutant strains BL21-pET-copA120H−N, BL21-pET-copA162H−N, BL21-pET-copA442H−N, BL21-pET-copA494H−N in LB medium. The culture conditions of the strain in Figure 4A were free of copper, and the strain in Figure 4B contained 0.25 mM Cu(Ⅱ). The reason why the manganese removal efficiency is negative is mainly the evaporation of water. By weighing the daily quality of the medium, it was found that its quality would decrease by 0.8 g to 1.7 g in one day. In the negative control group containing no microorganisms, the manganese concentration was increased by 10.4% after 8 days of culture. This should be due to evaporation of water. In addition, the growth and metabolism of bacteria also require water, so manganese removal efficiency has negative value. BL21-pET-copA120H−N and BL21-pET-copA494H−N exhibit manganese oxidizing ability faster than BL21-pET-copA162H−N and BL21-pET-copA442H−N. All strains showed maximum manganese oxidation rate from day 3 to day 6. With the increase of culture time, although 10 mM Tris-HCl(pH 7.0) was added to the system, the pH value of the culture system was gradually increased. Microorganisms also increase manganese oxidation efficiency by changing the pH of the system. In contrast, in the reaction system of CopA, the pH does not change. On the 8th day, the Mn(Ⅱ) removal efficiency of BL21-pET-copA494H−N reached 91.5%, and the Mn(Ⅱ) removal efficiency of the other strains was 89.9% (BL21-pET-copA), 70.1%(BL21-pET-copA120H−N), 87.1%(BL21-pET-copA162H−N), 84.4%(BL21-pET-copA442H−N), respectively(Table S2). Interestingly, comparing the two graphs 4A and 4B, the manganese removal efficiency of the strains was greatly reduced under the condition without Cu(Ⅱ). The manganese removal efficiency of each strain was 55.60% (BL21-pET-copA), -4.16% (BL21-pET-copA120H−N), 47.5% (BL21-pET-copA162H−N),57.5% (BL21-pET-copA442H−N), and 50.2% (BL21-pET-copA494H−N), respectively(Table S2).
The manganese removal efficiency of the mutant strains was inconsistent, indicating that the four conserved copper ion binding sites have different effects on the manganese oxidation process. By comparing the removal efficiency of two conditions, it is known that the Cu(Ⅱ) greatly promotes the manganese oxidation activity of strains. Interestingly, the manganese removal efficiency of strain BL21-pET-copA120H−N was the lowest in both copper-containing and copper-free conditions, indicating that H120 is the catalytically active site of the multicopper oxidase CopA.
Compared with the strain BL21-pET-copA, the other mutant strains showed no significant increase or decrease in manganese removal efficiency, indicating that the HXH (H means histidine; X means other amino acids) structure of the conserved copper binding site is coordinated and coordinated to maintain the stability of the oxidized structure. In addition, a mutant strain BL21-pET-copA494H−N with higher manganese oxidation activity was obtained in this study.
Global analysis of the LC-MS/MS results
The mass spectrometry data of CopA under three different conditions(condition a: multicopper oxidase CopA produced by strain BL21-pET-copA under copper-containing conditions; condition b: multicopper oxidase CopA produced by strain BL21-pET-copA under copper-free condition; condition c: multicopper oxidase CopA produced by mutant strain BL21-pET-copA120H-N under copper-free condition) were introduced into the software for analysis, the modification information of each sample was obtained.
There are overall 165 modifications in different peptides, and the specific modification information can be found in the supporting information(Table S3).
The main modification are carbamidomethyl, oxidation, deamidated, methyl, carboxymethyl, carbofuran, acetyl, ammonium, propionyl, gly, formyl, ethoxyformyl, in addition to sulfide, quinone, amidine, dihydroxyimidazolidine, propionamide, homocysteic acid, carbamyl, diethylphosphate modification. The sample of condition a has 117 modifications, the sample of condition b has 106 modifications, and the sample of condition c has 74 modifications. Comparison of the modification of sample a with sample b found that ethoxyformyl and quinone are modifications specific to sample a. Ethoxyformyl occurs on the histidine of the peptide MHLPGHSFTIVSTDGQPIHNPPETQDQLLNIAPGER. It is reported in the literature that when histidine is modified by ethoxyformyl, it will lose its ability to bind to metal ions, but will not change the conformation of the protein (Costa et al. 2009). Therefore, it is speculated that ethoxyformyl modifies the histidine of the peptide to allow Cu(II) to bind correctly to the histidine of the conserved copper ion binding sites.
Quinone is modified on the tryptophan of TAWTYNGTVPGPQLR. Quinone has a strong tendency to recover into a benzene ring structure and is highly oxidizing, so quinone is often used as an electron acceptor in oxidation reactions (Zengin et al. 2019). Therefore, it is speculated that the modification of quinone can increase the oxidation activity of CopA. The sample b was compared with the sample c, and no special modification was found, but the modification of sample c was greatly reduced. It has been reported that the presence of protein modifications can increase gene expression and is an important means of epigenetic regulation, so a reduction in modification results in incomplete structure and function of the protein (Veenstra 2003).
Peptide modification analysis
This section selects some peptides for detailed analysis. A total of 19 modifications were detected in the peptide MHLPGHSFTIVSTDGQPIHNPPETQDQLLNIAPGER, as shown in FIG 5A.The protein under a condition has 16 modifications, the protein under b condition has 10 modifications, and the protein under c condition has 10 modifications, mainly modified on histidine, threonine and methionine. Through the protein modification analysis of the same peptide segment, it can be seen that the change of the condition can reduce the modification of the amino acid, the position of the modification changes, and causing the activity of the protein decreases.
Since the multicopper oxidase CopA is an oxidizing enzyme, the peptides MPGHDMSK and MPGHDMSKMDSASTAEHENLK with more oxidation modifications were selected for analysis, as shown in FIG 5B.
Oxidation modifications in the peptide occur on methionine, and oxidative modification on methionine protects methionine from the dual effects of irreversible oxidative modification and regulation of protein function. This modification allows the protein to exert physiological functions under appropriate conditions, and can act as an antioxidant against various oxidative stresses in the environment. Under the conditions of a, b, and c, the sample a has more oxidation modification, indicating that under condition a, the protein has stronger oxidative resistance, can better protect the activity of the protein, and can avoid irreversible oxidation modification. Therefore, Cu(II) in culture conditions can increase the oxidation activity of protein CopA, which is consistent with the experimental results in 3.1.
Predicting the secondary structure of the protein before and after the mutation, it was found that the mutation of H120 caused its secondary structure to change, and the IT in the ITKY sequence was more obvious, as shown in the FIG S2. This sequence is present in the peptide sequence obtained by mass spectrometry, and therefore the peptides SESMDLPVVDITK and SESMDLPVVDITKYGEAAK containing this sequence are analyzed, as shown in Fig. 5C. It was found that the mutated protein, ie, the protein under the condition of c, had only one modification, and the other four modifications were lost due to the mutation, indicating that the mutation of the conserved copper binding site H120 would result in the loss of protein modification and affect the gene expression of the protein making the structure and function of the protein incomplete. The manganese oxidation activity of the H120 mutant strain was significantly reduced, which indicated that the protein modification of the multicopper oxidase CopA played an indispensable role in the manganese oxidation process.
Characterization of produced BioMnOx
The BioMnOx formed by multicopper oxidase CopA and recombinant strain BL21-pET-copA were characterized. The oxide formed by CopA shows a uniform layered structure(FIG 6b). This is different from the granulated and irregular polyhedral BioMnOx produced by the multicopper oxidase CueO (Su et al. 2014). It was found by the energy dispersive spectrometer(EDS) sweep these oxide that the oxidized particles were mainly composed of Mn and oxygen in the FIG S3.
The morphologies and structures of the oxide were further characterized with TEM. TEM confirm that the BioMnOx has a multi-layered mesh structure with irregularly distributed holes and many connection points (FIG 6e). In contrast, the analytical reagent-grade MnO2 particles were generally much larger crystalline particles and obviously needle structure(FIG 6c and 6f). The BioMnOx has a specific surface area and has a porous structure as compared with the chemically synthesized manganese oxide. Many studies have now used BioMnOx for the removal of heavy metal ions and organic contamination, and have achieved good results (Forrez et al. 2010; Sabirova et al. 2008).
SEM revealed finely grained, nanosize, amorphous, round manganese oxide particles produced by BL21-pET-copA (FIG 6a). Scanning these aggregates with EDS revealed that these aggregates were mainly composed of Mn, C and O elements in the FIG S3. Among them, C and O are the main elements of bacteria, and Mn and O are constituent elements of oxides. TEM observed that black small particles were formed on the surface of the cells, and as the manganese oxidation progresses, these particles become larger and larger, gradually covering the cell surface. (FIG 6d). This may be related to the physiology of microbial manganese oxidation, which oxidizes Mn(II) forming manganese oxide which surrounds the entire cell and forms a mesh-like structure. This can effectively protect cells from toxic substances, ultraviolet rays, reactive oxygen species, ionizing radiation, viruses and predation (Archibald and Fridovich 1981; Daly et al. 2004).
The valence electron configuration of the manganese atom is 3d5 4s2, so that polyvalent oxides (+2, +3, +4, +6, +7) can be formed. EDS can only determine the elemental composition of manganese oxide, but can’t measure its chemical valence. Therefore, the chemical valence of the BioMnOx were further determined by XPS. The XPS original map and fitting map of manganese oxides produced by CopA are shown in Fig. 7A. Four peaks were obtained by XPS peaking software with binding energies at 653.8 eV, 653.1 eV, 642.6 eV and 641.1 eV, respectively. The peaks of 653.8 and 642.6 are in agreement with MnO2; the peaks of 653.1 and 641.1 are in agreement with Mn3O4。The XPS original map and fitting map of manganese oxides produced by strain BL21-pET-copA are shown in Fig. 7B. Four peaks were obtained by XPS peaking software with binding energies at 653.8 eV, 653.1 eV, 642.2 eV and 641.1 eV, respectively. Given the literature comparison, the peaks of 653.8 and 642.2 are in agreement with MnO2; the peaks of 653.1 and 641.1 are in agreement with Mn3O4. Thus BioMnOx contain three different valence states, respectively Mn(II), Mn(III) and Mn(IV). By calculating the peak area, it is understood that the ratios of Mn(II), Mn(III), and Mn(IV) are 26.5%, 26.5%, and 47.0%, respectively, and the average oxidation state is 3.2. The average oxidation state of biological manganese oxides is generally lower than 4 because Mn(II) oxidation undergoes two successive one-step electron transfer processes, i.e., Mn(II) is first oxidized to Mn(III) and then Mn(III) oxidized to Mn (VI).