Pezizal halogenase pezw and uses thereof
By developing the phenazine halogenase PezW, the gap in the existing technology for the enzymatic preparation of hydroxylated phenazine compounds has been filled, realizing enzymatic halogenation modification and providing a new drug development pathway.
Patent Information
- Application Number
- CN202411884971.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2026-02-13
- Estimated Expiration
- 2044-12-19
AI Technical Summary
Current technologies have not yet enabled the enzymatic preparation of halogenated derivatives of hydroxylated phenazine compounds, while chemical synthesis methods pose environmental pollution problems.
A phenazine halogenase, PezW, derived from Streptomyces tubercidicus NBRC 13090, was developed. It is a flavin-dependent halogenase that can catalyze the transfer of halogen atoms to hydroxylated phenazine, thereby achieving enzymatic halogenation modification.
The enzymatic halogenation of hydroxylated phenazines was achieved, filling the technological gap in the enzymatic preparation of hydroxylated phenazines and providing a new pathway for the drug development of phenazine compounds, which has important application value.
Smart Images
Figure CN120683061B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of genetic engineering and biosynthesis, and relates to a halogenase, in particular to an enzyme for halogenation modification of hydroxyphenazine compounds. BACKGROUND
[0002] Halogen atoms have strong electronegativity, and their presence can improve the biological activity of compounds and change the physical and chemical properties of compounds. Therefore, the application of halogen elements in drugs, pesticides and new materials has attracted widespread attention from the scientific and industrial communities. Due to the environmental pollution and other problems in the chemical synthesis of halogenated organic compounds, halogenases in the biosynthesis pathway of natural halides have attracted more and more attention from scientists. Halogenases are enzymes that can catalyze halogen ions to form stable carbon-halogen bonds. The halogenases discovered so far are mainly divided into four types according to their mechanism and coenzyme (cofactor): haloperoxidase, alpha-ketoglutarate-dependent halogenase, flavin-dependent halogenase and S-adenosyl methionine-dependent fluorination enzyme. Among them, most halogenases use chlorine, bromine or iodine as halogen donors, with chlorine being the most common; only S-adenosyl methionine-dependent halogenase is a fluorination enzyme, which only uses fluorine as a halogen donor.
[0003] Phenazines are nitrogen-containing aromatic heterocyclic antibiotics of microbial origin. More than 100 phenazine natural products have been discovered so far, and they have broad-spectrum antibacterial and bacteriostatic activity. The diversity of natural phenazine compounds is mainly realized through various post-modification enzymes. Gebhardt et al. isolated isoprenylated modified phenazine derivatives Endophenazines A-D from the metabolites of Streptomycetes anulatus, among which Endophenazine A and Endophenazine D have good antibacterial activity. Wang et al. also isolated a phenazine and polyketide hybrid compound, Nexphenazine A, from Streptomyces sp. KIB-H483. In addition, more complex phenazine compounds composed of halogenated, oxymethyl, ester, amino, glycosyl and other substituents of phenazine have been found in metabolites of Streptomyces and Pseudomonas, among which there are effective substances with antibacterial and antitumor activities.
[0004] Among them, halogenated phenazines (HPs) exhibit significant antibacterial activity. So far, eight halogenated phenazines have been isolated and identified from natural products, including seven chlorinated and brominated products of terpene phenazines and one brominated product of a hydroxylated phenazine (2-bromo-1-hydroxyphenazine). Among them, 2-bromo-1-hydroxyphenazine exhibits significant antibiofilm activity against Staphylococcus aureus and Staphylococcus epidermidis, with a MIC value of 6.25 μM. Moreover, according to literature reports, halogenated derivatives of various hydroxylated phenazines (HHPs) exhibit significant antibiofilm activity.
[0005] At present, halogenated derivatives of hydroxylated phenazines are mainly obtained by direct fermentation and chemical total synthesis, and there is no report on the preparation of HHPs by enzyme method. Therefore, the halogenation modification of HHPs prepared by enzyme method will provide a new path for the development of phenazine compounds as drugs, and has important application value and social significance. SUMMARY
[0006] In order to fill the technical blank of the enzyme method for preparing halogenated derivatives of hydroxyl phenazine compounds in the prior art, the present application discloses a phenazine halogenase PezW for the first time. The halogenase PezW is derived from Streptomyces tubercidicus NBRC 13090 and belongs to flavin-dependent halogenase. The halogenase PezW can obtain various halogenated hydroxyl phenazines by taking hydroxyl phenazine as a substrate, which lays a foundation for the application of phenazine compounds in the development of drugs and has important practical application value.
[0007] Technical scheme of the present application:
[0008] A phenazine halogenase PezW, wherein the amino acid sequence of the phenazine halogenase PezW is selected from (1), (2) or (3):
[0009] (1) the amino acid sequence shown in SEQ ID NO: 1;
[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 and capable of catalyzing the transfer of halogen atom to hydroxyl phenazine;
[0011] (3) an amino acid sequence with a homology of ≥ 90% to the amino acid sequence shown in SEQ ID NO: 1, and the expressed protein is capable of catalyzing the transfer of halogen atom to hydroxyl phenazine.
[0012] The present application discloses phenazine halogenase PezW for the first time, realizes enzymatic halogenation of hydroxylated phenazine, fills the technical blank of enzymatic preparation of hydroxylated phenazine, and produces remarkable technical effects.
[0013] The DNA molecule encoding the phenazine halogenase PezW has a nucleotide sequence selected from the following (1), (2), (3) or (4):
[0014] (1) the nucleotide sequence shown in SEQ ID NO: 2;
[0015] (2) a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 2, but encoding the amino acid sequence shown in SEQ ID NO: 1;
[0016] (3) a nucleotide sequence with homology of ≥ 85% to the nucleotide sequence shown in SEQ ID NO: 2, and the expressed protein thereof can catalyze the transfer of a halogen atom to a hydroxylated phenazine;
[0017] (4) a nucleotide sequence complementary to the nucleotide sequence of any one of (1), (2) or (3).
[0018] An expression vector containing the nucleotide sequence encoding the phenazine halogenase PezW. The expression vector is a vector suitable for expression in E. coli.
[0019] An isolated recombinant vector containing the expression vector of the nucleotide sequence encoding the phenazine halogenase PezW.
[0020] A host cell comprising the isolated recombinant vector as described above; the host cell does not include animal and plant varieties.
[0021] A cloning and expression method of the phenazine halogenase PezW as described above, comprising the following steps: cloning the nucleotide sequence encoding the phenazine halogenase PezW into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for expression; and finally purifying to obtain the phenazine halogenase PezW.
[0022] The phenazine halogenase PezW as described above is applied in the preparation of a halogen-modified compound. The compound is a hydroxylated phenazine, specifically 1-hydroxyphenazine, 2-hydroxyphenazine or 1,6-dihydroxyphenazine. The halogen modification is the transfer and combination of a halogen atom of a halogen donor to a phenazine compound; the halogen donor is NaCl, NaBr or NaI.
[0023] Preferably, the reaction system of the enzyme catalyzed reaction is: 50 mM HEPES buffer (pH 7.4), 10 mM halogen donor, 0.5 mM hydroxylated phenazine, 100 μM FAD, 5 μM PezW protein, 1 mM NADH, ddH2O 27 μL; placed in a 30°C water bath reaction for 4h.
[0024] Advantages of the present application:
[0025] (1) The present application provides a phenazine halogenase PezW, thereby realizing the enzymatic halogenation of hydroxylated phenazine, filling the technical gap of enzymatic preparation of hydroxylated phenazine, and producing significant technical effects.
[0026] (2) The phenazine halogenase PezW described in the present application realizes the transfer of halogen atoms (Cl - , Br - , I - ) to hydroxylated phenazine substrates, which has a milestone significance for the development of enzyme synthesis of HHPs technology.
[0027] (3) The phenazine halogenase PezW described in the present application can halogenate and modify hydroxylated phenazine through enzyme catalysis, providing a new path for the drug research of phenazine compounds, and has important application value and social significance. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 is an SDS-PAGE analysis electrophoretogram of the phenazine halogenase PezW purified in Example 1 of the present application; Figure 1 Figure 2 is a high performance liquid chromatography (HPLC) chart of the system after the reaction of the phenazine halogenase PezW with 1-hydroxyl phenazine in Example 2-4 of the present application;
[0029] Figure 2 Figure 3 is a UV absorption chart of the substrate and product after the reaction of the phenazine halogenase PezW with 1-hydroxyl phenazine in Example 2-4 of the present application;
[0030] Figure 4 is a high performance liquid chromatography (HPLC) chart of the system after the reaction of the phenazine halogenase PezW with 1,6-dihydroxyl phenazine in Example 5-7 of the present application; Figure 3 Figure 5 is a UV absorption chart of the substrate and product after the reaction of the phenazine halogenase PezW with 1,6-dihydroxyl phenazine in Example 5-7 of the present application;
[0031] Figure 4 Figure 6 is a UV absorption chart of the substrate and product after the reaction of the phenazine halogenase PezW with 1,6-dihydroxyl phenazine in Example 5-7 of the present application;
[0032] Figure 7 is a UV absorption chart of the substrate and product after the reaction of the phenazine halogenase PezW with 1,6-dihydroxyl phenazine in Example 5-7 of the present application; Figure 5 Figure 8 is a UV absorption chart of the substrate and product after the reaction of the phenazine halogenase PezW with 1,6-dihydroxyl phenazine in Example 5-7 of the present application;
[0033] Figure 6 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1-hydroxyphenoxazinone in the presence of NaCl as halogen donor in the present application in Example 2;
[0034] Figure 2 Figure 7 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1-hydroxyphenoxazinone in the presence of NaBr as halogen donor in the present application in Example 3;
[0035] Figure 3 Figure 8 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1-hydroxyphenoxazinone in the presence of NaI as halogen donor in the present application in Example 4;
[0036] Figure 4 Figure 9 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1,6-dihydroxyphenoxazinone in the presence of NaCl as halogen donor in the present application in Example 5;
[0037] Figure 5 Figure 10 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1,6-dihydroxyphenoxazinone in the presence of NaBr as halogen donor in the present application in Example 6;
[0038] Figure 6 Figure 11 High resolution mass spectrum (HR-MS) of the product of the reaction of phenoxazinone halogenase PezW with 1,6-dihydroxyphenoxazinone in the presence of NaI as halogen donor in the present application in Example 7.
[0039] Figure 7 Figure 12 Structures of the hydroxylated phenoxazinones and halogenated products of the halogenation reactions of Examples 2-7. DETAILED DESCRIPTION
[0040] The present application will be further described in conjunction with the examples below.
[0041] The raw materials and chemical reagents used in the examples of the present application were all purchased through commercial channels. Among them, the HEPES buffer was purchased from Shanghai McLean Biological Reagent Co., Ltd.; NaCl, NaBr and NaI were purchased from Shanghai Sangon Biological Engineering Co., Ltd.; FAD (Flavin Adenine Dinucleotide) was purchased from Sigma-Aldrich Company; NADH (Nicotinamide adenine dinucleotide) was purchased from Sigma-Aldrich Company.
[0042] Example 1: Cloning and in vitro expression of halogenase PezW gene
[0043] 1. Synthesis of PezW gene
[0044] The nucleotide sequence of PezW (SEQ ID NO:2) was synthesized by Qingke Biotechnology Co., Ltd. and ligated into the pUC19 vector; the synthesized vector powder was dissolved in an appropriate amount of water and set aside for later use.
[0045] 2. Construction of protein expression vectors
[0046] The water-soluble vector was used as a template for PCR.
[0047] Design primer pairs:
[0048] P1: 5'- ctcgag gccgaagaggctcttgatgcgc-3' / P2:5'- catatg atggataacgaactgcgggacgac-3'
[0049] PCR reaction system:
[0050] 5 μL (50 pmol) each of primer pairs P1 and P2, 2 μL template, 50 μL (0.5 U / μL) 2× DNA polymerase, and distilled water to a final volume of 100 μL.
[0051] PCR conditions:
[0052] Promoter amplification conditions: denaturation at 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 28 cycles; 72℃ for 5 min. Functional gene amplification conditions: denaturation at 95℃ for 5 min; 95℃ for 30 s, 65℃ for 30 s, 72℃ for 1 min 30 s, 28 cycles; 72℃ for 5 min.
[0053] The amplified DNA fragments were digested using NdeI and XhoI restriction endonucleases, respectively, and cloned into plasmid pET-30a to construct plasmid pET-30a-PezW. This plasmid was then introduced into *E. coli* BL21(DE3) cells, and expression of the target protein was induced by isopropyl thiogalactoside (IPTG). The cells were cultured at 16°C for 16 hours, centrifuged to collect the cells, reconstituted with buffer (0.05M Tris-HCl, 0.5M NaCl, pH 7.5), and then ultrasonically disrupted. The resulting enzyme extract was purified by Ni column affinity purification and ultrafiltration concentration.
[0054] The enzyme extract was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are detailed below. Figure 1 .Depend on Figure 1 It can be seen that the protein prepared in this embodiment is PezW with a molecular weight of 59.9 kDa.
[0055] The enzyme extract was concentrated and replaced into buffer (0.025 M Tris-HCl, 0.02 M NaCl, 10% glycerol, pH 7.5) and stored at -80°C for later use.
[0056] Example 2: In vitro enzyme activity assay of PezW protein
[0057] (1) Preparation of enzyme activity reaction system (100 μL) :
[0058] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaCl: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein (prepared in Example 1): 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL.
[0059] (2) Halogenation reaction:
[0060] The enzyme activity reaction system prepared in step (1) was reacted at 30°C for 4 h. After the reaction was completed, 100 μL of methanol was added, vortexed for 5 min, centrifuged at 13,000 rpm for 20 min, and the precipitate was discarded to obtain the supernatant, which was used as a sample for in vitro enzyme activity assay and HR-MS detection.
[0061] Example 3: In vitro enzyme activity assay of PezW protein
[0062] The difference from Example 2 is that,
[0063] (1) Preparation of enzyme activity reaction system (100 μL) :
[0064] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaBr: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL
[0065] (2) Halogenation reaction: same as Example 2.
[0066] Example 4: In vitro enzyme activity assay of PezW protein
[0067] The difference from Example 2 is that,
[0068] (1) Preparation of enzyme activity reaction system (100 μL) :
[0069] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL
[0070] (2) Halogenation reaction: same as Example 2.
[0071] Example 5: In vitro enzyme activity assay of PezW protein
[0072] Except that Example 2 was different in that,
[0073] (1) Preparation of in vitro enzyme activity reaction system (100 μL):
[0074] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL
[0075] (2) Halogenation reaction: same as Example 2.
[0076] Example 6: In vitro enzyme activity assay of PezW protein
[0077] Except that Example 2 was different in that,
[0078] (1) Preparation of in vitro enzyme activity reaction system (100 μL):
[0079] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL
[0080] (2) Halogenation reaction: same as Example 2.
[0081] Example 7: In vitro enzyme activity assay of PezW protein
[0082] Except that Example 2 was different in that,
[0083] (1) Preparation of in vitro enzyme activity reaction system (100 μL):
[0084] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1,6-dihydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL
[0085] (2) Halogenation reaction: Same as in Example 2.
[0086] Example 8: HPLC analysis of halogenation reaction products in Examples 2-7
[0087] HPLC detection conditions: reversed-phase C18 column (size: 150×4.6mm, 5μm); column temperature: 30℃; elution conditions: 0-5 min equilibration: 70% phase A (distilled water + 0.1% formic acid) and 30% phase B (acetonitrile + 0.1% formic acid); 5-45 min linear elution: 70-0% phase A and 30-100% phase B; 45-50 min isocratic elution: phase A: 0%, phase B: 100%; detection wavelength: 280nm; flow rate: 1mL / min.
[0088] Appendix Figure 2 The HPLC results are for the systems following the reaction of phenazine halogenase PezW with 1-hydroxyphenazine in Examples 2-4. Figure 2 It was found that the HPLC chromatograms of the samples with inactivated PezW and 1-hydroxyphenazine only showed a peak of the substrate 1-hydroxyphenazine at a retention time of 16 min. However, the HPLC chromatograms of the samples containing PezW + 1-hydroxyphenazine + halogen donor showed new chromatographic peaks. Specifically, when the halogen donor was NaCl, three peaks appeared at retention times of 19.6 min, 20.7 min, and 21.2 min. When the halogen donor was NaBr, three peaks appeared at retention times of 21.2 min, 21.4 min, and 22 min. When the halogen donor was NaI, one peak appeared at a retention time of 23.7 min.
[0089] Appendix Figure 3 The HPLC results are for the systems following the reaction of phenazine halogenase PezW with 1,6-dihydroxyphenazine in Examples 5-7. Figure 3It was found that the HPLC chromatograms of the samples with inactivated PezW and 1,6-dihydroxyphenazine showed a peak for the substrate 1,6-dihydroxyphenazine only at a retention time of 17.8 min. However, the HPLC chromatograms of the samples containing PezW + 1,6-dihydroxyphenazine + halogen donor showed new chromatographic peaks. Specifically, when the halogen donor was NaCl, four peaks appeared at retention times of 22.1 min, 22.3 min, 25.5 min, and 26.0 min. When the halogen donor was NaBr, three peaks appeared at retention times of 23.2 min, 23.5 min, and 26.5 min. When the halogen donor was NaI, one peak appeared at a retention time of 25.0 min.
[0090] In summary, based on the HPLC results, different peaks were observed in systems using NaCl, NaBr, and NaI as halogen donors and 1-hydroxyphenazine and 1,6-dihydroxyphenazine as substrates. This indicates that the hydroxylated phenazine underwent a chemical reaction with the phenazine halogenase PezW, yielding different products.
[0091] Example 9: Ultraviolet Detection and Analysis of Halogenation Reaction Products in Examples 2-7
[0092] Ultraviolet detection conditions: Diode Array Detector (DAD), detection range 190-400nm.
[0093] Appendix Figure 4 The UV absorption spectra of the substrates and products of the reaction between phenazine halogenase PezW and 1-hydroxyphenazine in Examples 2-4 are shown. Figure 4 It can be seen that the UV absorption peak of compound 1 (1-hydroxyphenazine) is located at 270 nm, and the UV absorption peak of compound 1a-1g is also located at 270 nm. This indicates that compound 1a-1g is a product of halogenation modification based on compound 1.
[0094] Appendix Figure 5 The UV absorption spectra of the substrate and product of the reaction between phenazine halogenase PezW and 1,6-dihydroxyphenazine in Examples 5-7 of this invention are shown. Figure 5 It can be seen that the UV absorption peak of compound 2 (1,6-dihydroxyphenazine) is located at 280 nm, and the UV absorption peak of compounds 2a-2h is also located at 280 nm. This indicates that compounds 2a-2h are halogenated products based on compound 2.
[0095] Combined with the HPLC chromatogram of Example 8 ( Figures 2-3As can be seen, the reaction of 1-hydroxyphenazine / 1,6-dihydroxyphenazine with the phenazine halogenase PezW yields a halogenated product, which indicates the occurrence of an enzymatic reaction; thus confirming that the halogenase PezW provided in this application catalyzes the transfer of halogen atoms to the hydroxylated phenazine substrate.
[0096] Example 10: High-resolution mass spectrometry (HR-MS) analysis of halogenation reaction products in Examples 2-7
[0097] HR-MS analysis conditions: reversed-phase C18 column (size: 150×4.6mm, 5μm); column temperature: 30℃; elution conditions: 0-5 min equilibration: 95% phase A (distilled water + 0.1% formic acid) and 5% phase B (acetonitrile + 0.1% formic acid); 5-15 min linear elution: 95-5% phase A and 5-95% phase B; 15-18 min isocratic elution: phase A: 0%, phase B: 100%; detection wavelength: 280nm; flow rate: 0.3mL / min; ion source type: electrospray ionization (ESI); ion mode: positive ion mode; acquisition range: m / z: 200-1000Da.
[0098] Appendix Figure 6 The above are the HR-MS spectra of products 1a-1c from the reaction of phenazine halogenase PezW with 1-hydroxyphenazine (NaCl as the halogen donor) in Example 2. (According to Appendix...) Figure 6 It can be seen that when NaCl is used as a halogen donor and 1-hydroxyphenazine (1, [M+H] = 197.07) is used in the enzymatic reaction, the [M+H] of products 1a-1b is 231.03, and that of 1c is 264.99. Compared with the molecular weight of the substrate, the molecular weight of 1a-1b increased by 33.96 Da, confirming that 1a-1b is a monochloro product; the molecular weight of 1c increased by 67.92 Da, confirming that 1c is a dichloro product. The molecular weight is consistent with the expected molecular weight of the target product, indicating that the chlorination reaction occurred.
[0099] Appendix Figure 7 The image shows the HR-MS spectra of the reaction product 1d-1f of phenazine halogenase PezW and 1-hydroxyphenazine (NaBr as halogen donor) in Example 3. (According to the appendix...) Figure 7It can be seen that when NaBr is used as a halogen donor and 1-hydroxyphenazine (1, [M+H] = 197.07) is used in the enzymatic reaction, the [M+H] of products 1d-1e is 274.98 and that of 1f is 354.89. Compared with the molecular weight of the substrate, the molecular weight of 1d-1e increased by 77.91 Da, confirming that 1d-1e is a monobrominated product; the molecular weight of 1f increased by 155.82 Da, confirming that 1f is a dibrominated product. The molecular weight is consistent with the expected molecular weight of the target product, indicating that the bromination reaction has occurred.
[0100] Appendix Figure 8 The image shows the HR-MS spectrum of 1 g of the reaction product of phenazine halogenase PezW and 1-hydroxyphenazine (NaI as the halogen donor) in Example 4. (According to Appendix...) Figure 8 It can be seen that when NaI is used as a halogen donor and 1-hydroxyphenazine (1, [M+H] = 197.07) is used as the substrate for the enzymatic reaction, the [M+H] of 1g of product is 322.97. Compared with the molecular weight of the substrate, the molecular weight of 1g increases by 125.90 Da, confirming that 1g is a monoiodinated product. The molecular weight is consistent with the expected molecular weight of the target product, indicating that the iodination reaction has occurred.
[0101] Appendix Figure 9 The above are the HR-MS spectra of products 2a-2d from the reaction of phenazine halogenase PezW with 1,6-dihydroxyphenazine (NaCl as the halogen donor) in Example 5. (According to Appendix...) Figure 9 It can be seen that when NaCl is used as a halogen donor and the substrate is 1,6-dihydroxyphenazine (2, [M+H] = 213.07), the [M+H] of products 2a-2b is 247.02, and that of 2c-2d is 280.99. Compared with the molecular weight of the substrate, the molecular weight of 2a-2b increased by 33.96 Da, confirming that 2a-2b is a monochloro product; the molecular weight of 2c-2d increased by 67.92 Da, confirming that 2c-2d is a dichloro product. The molecular weights are consistent with the expected molecular weights of the target products, indicating that the chlorination reaction occurred.
[0102] Appendix Figure 10 The image shows the HR-MS spectrum of the reaction product 2e-2g of phenazine halogenase PezW and 1,6-dihydroxyphenazine (NaBr as halogen donor) in Example 6. (According to Appendix...) Figure 10It can be seen that when NaBr is used as a halogen donor and the substrate is 1,6-dihydroxyphenazine (2, [M+H]=213.07), the [M+H] of the product 2e-2f is 290.98, and the [M+H] of 2g is 370.88. Compared with the molecular weight of the substrate, the molecular weight of 2e-2f increases by 77.91 Da, confirming that 2e-2f is a monobrominated product; the molecular weight of 2g increases by 155.82 Da, confirming that 2g is a dibrominated product. The molecular weight is consistent with the molecular weight of the expected target product, indicating that the bromination reaction occurs.
[0103] Figure 8 is an HR-MS spectrum of the product 2h of the reaction of the phenazine halogenase PezW with 1,6-dihydroxyphenazine (NaI as a halogen donor) in Example 7. Figure 11 Figure 11 It can be seen that when NaI is used as a halogen donor and the substrate is 1,6-dihydroxyphenazine (2, [M+H]=213.07), the [M+H] of the product 2h is 338.96. Compared with the molecular weight of the substrate, the molecular weight of 2h increases by 125.90 Da, confirming that 1g is a monoidinated product. The molecular weight is consistent with the molecular weight of the expected target product, indicating that the iodination reaction occurs.
[0104] In summary, the halogenase PezW provided in the present application realizes the transfer of multiple halogen atoms from NaCl / NaBr / NaI to the hydroxyphenazine substrate; realizes the enzymatic halogenation of hydroxyphenazine, fills the technical gap of enzymatic preparation of hydroxyphenazine, and produces significant technical effects. This not only clears the obstacles for the enzymatic preparation of various HHPs, but also provides a new path for the drug research of phenazine compounds, which has important social significance and great application prospect.
Claims
1. Use of a phenoxazinum halogenase PezW for the preparation of a halogenated modified compound, characterized in that: The amino acid sequence of the phenoxazine halogenase PezW is shown as SEQ ID NO: 1, and the compound is a hydroxylated phenoxazine.
2. Use according to claim 1, characterized in that: The hydroxylated phenoxazine is specifically 1-hydroxyphenoxazine, 2-hydroxyphenoxazine or 1,6-dihydroxyphenoxazine.
3. Use according to claim 1, characterized in that: The halogenation modification is transferring and combining the halogen atom of a halogen donor to the phenoxazine compound; and the halogen donor is NaCl, NaBr or NaI.
4. Use according to any one of claims 1 to 3, characterized in that: The halogenation modification is modifying the ortho and para positions of the hydroxyl group in the hydroxylated phenoxazine through an enzyme catalytic reaction.
5. Use according to claim 4, characterized in that: The reaction system of the enzyme catalytic reaction is: 50 mM HEPES buffer pH 7.4, 10 mM halogen donor, 0.5 mM hydroxylated phenoxazine, 100 μM FAD, 5 μM PezW protein, 1 mM NADH, 27 μL of distilled water.