O-methyltransferases, genes, mutants and fusion enzymes and uses thereof

Abstract

The application discloses a kind of O Methyltransferase, gene, mutant and fusion enzyme and its application belong to the field of bioengineering technology.This application provides a kind of methyltransferase SmOMT O Methyltransferase SmOMT has substrate promiscuity and regional selectivity and its amino acid sequence is as shown in SEQ ID NO.1.Mutant SmOMT M2 Is obtained by carrying out E152A and I306A double mutation based on SmOMT, has higher catalytic activity.Meanwhile, the application constructs a SAM non-dependent methylation system, which includes mutant SmOMT M2 And mutant halide methyltransferase AtHMT V140T Formed fusion enzyme AtHMT V140T -L 95 -SmOMT M2 The fusion enzyme can realize the methylation of various substrates without exogenous SAM, and the catalytic efficiency is higher than that of free enzyme level system, and can be used for synthesizing methylated compounds with enhanced anti-inflammatory activity.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a... O 1-Methyltransferases, genes, mutants and fusion enzymes and their applications. Background Technology

[0002] The methyl group is one of the most common substituents in drug molecules, second only to hydrogen and nitrogen. According to statistics from Njardarson's research group, more than half of the top 200 best-selling small molecule drugs globally in 2023 contained at least one methyl group. Despite their small size, the methyl group has a profound impact on the molecular properties of drugs. Strategically introducing methyl groups can enhance biological activity, regulate metabolic stability, alter solubility, improve target selectivity, and in some cases, even convert agonists into antagonists. Medicinal chemists call this phenomenon the "methyl magic effect." For example, methylating the abandoned compound mevastatin yielded the non-carcinogenic drug lovastatin. Further methylation produced simvastatin, which has twice the half-life of lovastatin, enhanced efficacy, and annual sales of $5.6 billion. Similarly, α-methylation of the proline residues of Glypromate yielded trafinone, which exhibited an extended plasma half-life and improved oral bioavailability, overcoming the drawbacks of rapid metabolism and poor delivery of the original drug, thus gaining approval from the U.S. Food and Drug Administration (FDA) for the treatment of Rett syndrome.

[0003] Furthermore, methylation is ubiquitous in the biosynthesis of natural products (NPs), playing a crucial role in their structural diversity and biological activity. In plants, methylation is catalyzed by S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs; EC 2.1.1.). Based on the type of atom targeted by methylation, methyltransferases can be classified into C–, O–, N–, and S– methyltransferases. O α-methyltransferase ( O O-methyltransferases (OMTs) constitute the largest and most functionally diverse category, and are therefore the most extensively studied. Compared to chemical synthesis, enzymatic O-methylation has significant advantages. It enables precise regioselective and stereoselective modification in complex substrate mixtures, thus avoiding expensive multi-step protection-deprotection strategies and eliminating dependence on toxic methylating agents. Furthermore, enzymatic O-methylation is a green catalytic method, carried out under mild conditions and with high yields, making it an ideal platform for synthesizing methylated bioactive molecules.

[0004] In recent years, an increasing number of plant OMTs (O-methyltransferases) with well-defined functions have been identified and used for the enzymatic synthesis of natural products. Despite significant progress in the identification and characterization of plant OMTs, research has primarily focused on the methylation of flavonoids, alkaloids, and coumarins. Most reported plant OMTs exhibit strict substrate selectivity and a narrow substrate range, limiting their application in the catalytic synthesis of other structurally diverse natural products. OMTs with substrate heterogeneity are generally considered promising catalysts for the methylation of small molecules. However, only a few OMTs with moderate substrate heterogeneity have been identified so far. Structural studies have shown that plant OMTs methylate via S... N Type II nucleophilic substitution mechanisms catalyze MT-mediated methylation. However, the structural basis for the observed substrate heterogeneity and regioselectivity remains poorly understood. Large-scale application of OMTs is also hampered by two major limitations: the high cost and limited supply of SAMs; and the potent product inhibition caused by the accumulation of S-adenosyl-L-homocysteine ​​(SAH) after methyl transfer. Although in vivo and in vitro SAM regeneration systems have been developed, the significant decrease in methylation efficiency due to interference between multiple free enzymes in these systems remains a persistent challenge. Summary of the Invention

[0005] This invention screened SmOMT, a methyltransferase from Selaginella tamariscina, and obtained its encoding gene. This Selaginella tamariscina methyltransferase can catalyze the methylation of caffeic acid, acetophenones, flavonoids, or coumarins, etc., with a very broad substrate range.

[0006] This invention provides an O-methyltransferase SmOMT, whose amino acid sequence is SEQ ID NO.1. SEQ ID NO.1 specifically is...

[0007] MGSAGGVIVEDEDRLQIMELATMCSVPMALKVAVEMDVAERIEKAGPGGLLSAAEIVSQIPECSSPMSPIYLDRIMRVLASRKIFKEVDEGGVRKYGLTSMCKHLIKDERGVSLAHHVLMNQDKVFMETWQYLHEAVLDGGEPFTKAFGQTEFELGKENSRVNNLFHAAMSNHSKLY MNAILEAYHGFKGIGTLVDVGGGVGTSLTVILKKYPEIKGINFDLPHVVAKAPQIPGVEHVGGDMFVSVPQGDAIFMKWILHDWSDEACITLLKNCYKSIPEHGKVIVVDSVLPSVLDTGAGARVALSIDLLMLVYNPGGKERTFEDFEKLAKASGFSSVKVPVTVDFISVVEFHK.

[0008] This patent marks the first identification of a unique O-methyltransferase, SmOMT, from the medicinal plant *Selaginella moellendorffii*. This enzyme exhibits significant substrate heterogeneity, catalyzing the methylation of at least 25 structurally diverse substrates, encompassing various types of compounds including flavonoids, coumarins, acetophenones, resveratrol, and anthraquinones. Furthermore, SmOMT possesses specific regioselectivity, enabling precise methylation modification at specific sites on the substrate, thus providing a highly efficient and multifunctional biocatalyst for methylation reactions.

[0009] The nucleotide sequence of the SmOMT gene is SEQ ID NO.3. SEQ ID NO.3 specifically is:

[0010]

[0011] This invention provides an O-methyltransferase mutant, SmOMT. M2 It was obtained by double mutation of E152A and I306A based on the aforementioned SmOMT.

[0012] This patent is based on in-depth structural analysis of SmOMT and modifies it using site-directed mutagenesis technology to obtain a double mutant SmOMT. M2 (E152A / I306A). Compared to wild-type SmOMT, this mutant exhibits significantly enhanced catalytic activity, increasing by 2.6 times, enabling more efficient catalysis of methylation reactions of various substrates and further enhancing the enzyme's application potential in biocatalysis. Furthermore, the single mutant E152A also demonstrates high catalytic activity, increasing by 2.3 times compared to wild-type SmOMT.

[0013] Correspondingly, this patent protects the aforementioned SmOMT. M2 The gene encoding E152 and I306 was obtained by mutating the nucleotides encoding E152 and I306 based on SEQ ID NO.3 (specifically, the gene mutation results in the 152nd amino acid changing from E to A and the 306th amino acid changing from I to A).

[0014] This patent provides a fusion enzyme (AtHMT) V140T –L 95 –SmOMT M2 ), from the aforementioned SmOMT M2 With mutant halide methyltransferase AtHMT V140T Through L 95 It is formed by connecting (connector 95), and its amino acid sequence is SEQ ID NO.2. To construct a more efficient methylation system, this patent uses SmOMT... M2 With mutant halide methyltransferase AtHMT V140T Through L 95 The AtHMT fusion enzyme was successfully designed and constructed. V140T –L 95 –SmOMT M2 This fusion enzyme exhibits higher efficiency than the free enzyme cascade system in catalyzing the methylation of various substrates (such as compounds 2, 3, 11, 16, 17, and 27). This is mainly due to the synergistic effect between enzyme molecules after fusion, which reduces interference between free enzymes and thus improves the overall catalytic efficiency.

[0015] Specifically, L 95 AtHMT V140T N-terminus and SmOMT M2is linked to the C-terminus. SEQ ID NO.2 is specifically: MAEEQQNSDQSNGGNVIPTPEEVATFLHKTVEEGGWEKCWEEEITPWDQGRATPLIVHLVDTSSLPLGRALVPGCGGGHDVVAMASPERFVVGLDISESALAKANETYGSSPKAEYFSFVKEDVFTWRPTELFDLIFDYTFFCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHVGGPPYKVDVSTFEEVLVPIGFKAVSVEENPHAIPTRKGKEKLGRWKKINPTPTPPTPTPGSAGGVIVEDEDRLQIMELATMCSVPMALKVAVEMDVAERIEKAGPGGLLSAAEIVSQIPECSSPMSPIYLDRIMRVLASRKIFKEVDEGGVRKYGLTSMCKHLIKDERGVSLAHHVLMNQDKVFMETWQYLHEAVLDGGEPFTKAFGQTAFELGKENSRVNNLFHAAMSNHSKLYMNAILEAYHGFKGIGTLVDVGGGVGTSLTVILKKYPEIKGINFDLPHVVAKAPQIPGVEHVGGDMFVSVPQGDAIFMKWILHDWSDEACITLLKNCYKSIPEHGKVIVVDSVLPSVLDTGAGARVALSADLLMLVYNPGGKERTFEDFEKLAKASGFSSVKVPVTVDFISVVEFHK.

[0016] V140T The gene encoding L 95 The gene and encoding SmOMT M2 The genes, and these three genes are connected in sequence.

[0017] This patent provides a recombinant vector comprising SmOMT and SmOMT. M2 or fusion enzyme AtHMT V140T –L 95 –SmOMT M2 Furthermore, this patent provides a recombinant host cell containing the aforementioned recombinant vector or carrying SmOMT, SmOMT gene. M2 or fusion enzyme AtHMT V140T –L 95 –SmOMT M2 The genes.

[0018] These vectors and host cells provide effective tools for the expression and production of related enzymes, which helps to promote the application of this methylation system in actual production.

[0019] This patent also provides O-methyltransferase SmOMT and O-methyltransferase mutant SmOMT. M2 or fusion enzyme AtHMT V140T –L 95 –SmOMT M2 In the application of catalytic methylation reactions, the substrates for methylation reactions are selected from flavonoids (compounds 2, 3, 6, 8-11, etc.), coumarins (compounds 12-17, etc.), acetophenones (compounds 19, 21, 22, 24, 25, 27, 28, 31, 32, etc.), resveratrol (compound 33), or anthraquinone compounds (compound 34), etc.

[0020] For O-methyltransferase SmOMT and O-methyltransferase mutant SmOMT M2Using SAM as the methyl donor and caffeic acid as the acceptor as an example, the conversion rate was greater than 60% under reaction times greater than 140 min, reaction temperatures of 30-50 °C, and pH 8.0 (50 mM Tris-HCl) or pH 7.0 (50 mM Na2HPO4-NaH2PO4). The optimized conditions were: under pH 8.0 (50 mM Tris-HCl) or pH 7.0 (50 mM Na2HPO4-NaH2PO4) conditions, after treatment at 37 °C for 4 hours, the SmOMT enzyme activity reached its peak, and this enzyme activity was independent of divalent metal ions. Specifically, for the catalysis of flavonoids, SmOMT tends to methylate the 3'-OH group of flavonoids. For acetophenone derivatives, SmOMT has a strong preference for the meta-hydroxyl group in acetophenone compounds.

[0021] Specifically, see Figure 4 The SmOMT exhibits very high catalytic activity for compounds 1, 6, 12, 13, 24, 25, 28, 31, 32 and 34.

[0022] For fusion enzymes, compounds 2, 3, 8, 11, 12, 16, 17, 27 and 31, using CH3I and SAH as methyl donors, all exhibit catalytic activity. Among them, compounds 2, 16, 27 and 31 all have very high catalytic activity.

[0023] This patent also provides the application of the fusion enzyme in SAM-independent methylation. The methylation process is as follows: CH3I and SAH are used as methyl donors. Taking luteolin (compound 2) as an example, the conversion rate is greater than 60% under the following conditions: reaction time greater than 120 min, reaction temperature 25-45℃, pH 6.0 (50mM Na2HPO4–NaH2PO4 buffer), and CH3I / SAH molar ratio of 1:1-5:1. The optimized conditions are: under the following conditions: pH 6.0 (50mM Na2HPO4–NaH2PO4 buffer), CH3I / SAH molar ratio of 2, after treatment at 37°C for 3 hours, the enzyme activity of the fusion enzyme reaches its peak.

[0024] This patent uses the fusion enzyme AtHMT. V140T –L 95 –SmOMT M2 As a catalyst, CH3I and SAH can be used as raw materials to carry out methylation reactions under suitable reaction conditions without the need for exogenous SAM, thus overcoming the dependence of traditional enzymatic methylation on expensive SAM.

[0025] Experiments have shown that this fusion enzyme has broad applications in catalyzing methylation reactions, acting on a variety of substrates, including flavonoids, coumarins, acetophenones, resveratrol, and anthraquinones. This fusion enzyme system has successfully synthesized a variety of methylated compounds, and assays using the RAW264.7 cell model revealed that the anti-inflammatory activity of these products (such as compounds 2, 8, 12, and 31) was significantly enhanced compared to their parent compounds. Attached Figure Description

[0026] Figure 1 The electrophoresis results of amplifying the target gene fragment SmOMT using cDNA from Selaginella tamariscina as a template are shown.

[0027] Figure 2 SDS-PAGE gel electrophoresis image and molecular sieve image of purified SmOMT;

[0028] Figure 3 This is a line graph showing the reaction time, reaction temperature, pH, and metal ions of SmOMT in this invention.

[0029] Figure 4 The graph shows the conversion rates of compounds in the SmOMT substrate miscibility experiment.

[0030] Figure 5 HPLC and LC-MS spectra of 25 substrates;

[0031] Figure 6 For product 2a 1 H spectrum and 13 C spectrum;

[0032] Figure 7 For product 6a 1 H spectrum and 13 C spectrum;

[0033] Figure 8 For product 8a 1 H spectrum and 13 C spectrum;

[0034] Figure 9 For product 9a 1 H spectrum and 13 C spectrum;

[0035] Figure 10 For product 10a 1 H spectrum and 13 C spectrum;

[0036] Figure 11 For product 24a 1 H spectrum and 13 C spectrum;

[0037] Figure 12 For product 31a 1 H spectrum and 13 C spectrum;

[0038] Figure 13 For product 32a 1 H spectrum and 13 C spectrum;

[0039] Figure 14 The relative conversion rate of SmOMT mutant and SmOMT E152A / I306A Conversion plots for other substrates;

[0040] Figure 15 The diagram shows the SmOMT-AtHMT coupling reaction mode and the HPLC chromatogram using 31 as the substrate.

[0041] The top left image shows the schematic diagram of SmOMT-AtHMT catalysis; the top right image shows the HPLC chromatogram using compound 31 as a substrate; the bottom left image compares the catalytic effects of the free enzyme cascade system with different connector systems; and the bottom right image shows the catalytic effects of compounds 2, 3, 11, 16, 17, and 27 in the free enzyme cascade system and AtHMT. V140T –L 95 –SmOMT M2 A comparison chart of catalytic activities;

[0042] Figure 16 Line graphs showing reaction time, reaction temperature, pH, and CH3I / SAH ratio for the fusion enzyme system;

[0043] Figure 17 A comparison chart of the anti-inflammatory activities of 2 / 2a, 8 / 8a, 12 / 12a, and 31 / 31a. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0045] Example 1: O Cloning and Construction of Expression Vector for β-Methyltransferase SmOMT

[0046] 1. The whole plant of *Selaginella tamariscina* was flash-frozen in liquid nitrogen and ground into a fine powder. RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441, purchased from Tiangen Biotech (Beijing) Co., Ltd.). The obtained RNA sample was used to synthesize cDNA using the ABScript III RT Master Mix for qPCR with gDNA Remover (RK20429, purchased from Wuhan Aibote Biotechnology Co., Ltd.). PCR amplification was performed using the synthesized cDNA as a template. Primer information is as follows:

[0047] Upstream primer (SmOMT1-F):

[0048] 5' GTGCCGCGCGGCAGCCATATGATGGAGGACGAGAGCTGGATG 3'

[0049] Downstream primer (SmOMT1-R):

[0050] 5' CTCGAGTGCGGCCGCAAGCTTTCAAGGGTATATCTCGATGATGTATGC 3'

[0051] The amplification system consisted of 25 μl of 2×PhantaMax Buffer, 1 μl of dNTP Mix (10 mM), 2 μl of cDNA, 2 μl each of upstream and downstream primers, and Nuclease-free H2O to a final volume of 50 μl. The PCR amplification program was as follows: ① 95℃ for 3 min; ② 95℃ for 15 s, 60℃ for 15 s, 72℃ for 60 s, for a total of 30 cycles; ③ 72℃ extension for 5 min. After the reaction, the product size was determined by 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown.

[0052] 2. Construction of recombinant plasmids

[0053] The amplified product and the linearized pET28a(+) vector were homologously cloned using the Hieff Clone Plus Multi One Step Cloning Kit. The reaction mixture was then transferred into competent DH5α cells, and the reaction products were sent to a sequencing company for sequencing.

[0054] Example 2: O Expression and purification of β-methyltransferase SmOMT

[0055] The recombinant plasmid pET28a(+)-SmOMT was transformed into *E. coli* Rosetta (DE3) for heterologous expression. Transformants were screened on LB agar plates containing 50 μg / mL kanamycin. Single colonies were picked and inoculated into 80 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm with shaking. The culture was then transferred to 8 L of LB medium containing 50 μg / mL kanamycin. Cells were cultured at 37°C and 220 rpm until OD (digestive activity) was achieved. 600 The target protein concentration was 0.6-0.8. Protein expression was induced by adding 0.5 mM IPTG and inducing at 18°C ​​and 180 rpm for 20 h. Cells were collected by centrifugation at 4°C and 6000 rpm for 10 min. The pellet was resuspended in 100 mL of lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). Cells were homogenized using a high-pressure homogenizer. The lysis buffer was centrifuged at 4°C and 12000 rpm for 45 min, and the supernatant containing the target protein was collected and loaded onto a Ni-NTA column pre-equilibrated with lysis buffer. The target protein was eluted using elution buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 300 mM imidazole). The fractions containing the target protein were combined and concentrated to 2 mL using an Amicon Ultra-10k centrifuge filter (Merck Millipore). The concentrated solution was loaded onto a HiPrep 16 / 60 Sephacryl S-200HR column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. Protein purity was confirmed by SDS-PAGE electrophoresis. The purified protein was concentrated to 10 mg / mL and stored at -80°C. The results are as follows: Figure 2 As shown.

[0056] Example 3: Enzymatic Properties Analysis of SmOMT1

[0057] The enzymatic properties of SmOMT were characterized, and its optimal reaction time, pH, temperature, and divalent metal ion dependence were determined. Eleven time points were selected within a reaction time range of 5 to 360 minutes for evaluation. For pH, four buffer systems were used for enzyme activity assays: citrate-sodium citrate (pH 3.0–6.0), Na₂HPO₄ / NaH₂PO₄ (pH 6.0–8.0), Tris-HCl (pH 6.0–8.0), and Na₂CO₃ / NaHCO₃ (pH 9.0–11.0). Temperature dependence was tested between 4°C and 60°C. The metal ion effect was assessed by detecting Ba. 2+ Fe 2+ Ca 2+Mg 2+ The concentration gradient of EDTA and a blank control were used to investigate the effects. All reactions used SAM as the methyl donor and caffeic acid as the acceptor, and each experiment was repeated three times. The reaction was terminated by adding methanol, followed by HPLC analysis after centrifugation at 12000 rpm for 20 minutes.

[0058] HPLC detection conditions were: Thermo Ultimate 3000; XSelect® HSS T3 5μm column; acetonitrile-formic acid water (0.1%); 0-20 min 10%-100% acetonitrile, 20-25 min 100% acetonitrile, 25-28 min 100%-10% acetonitrile, 28-35 min 10% acetonitrile; 0.8 mL / min. Results are as follows: Figure 3 As shown.

[0059] Biochemical characterization showed that SmOMT enzyme activity reached its peak after treatment at 37°C for 4 hours under pH 8.0 (50 mM Tris-HCl) or pH 7.0 (50 mM Na2HPO4-NaH2PO4) conditions. This enzyme activity was independent of divalent metal ions, as shown in the results. Figure 3 As shown.

[0060] Example 4: Substrate heterogeneity analysis and preparative-scale reaction of SmOMT

[0061] The substrate heterogeneity of SmOMT was investigated using 34 substrates (numbered 1-34). The 100 μL reaction mixture used for enzyme activity assays contained 0.5 mM substrate, 2 mM SAM, 50 mM Tris-HCl (pH 8.0), and 100 μg SmOMT. The reaction was incubated overnight at 37 °C, terminated by adding 100 μL of methanol, and then centrifuged at 12000 rpm for 20 min. The supernatant was analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC / MS), with all reactions performed in triplicate. The preparative-scale reaction (final volume 20 mL) contained 50 mM Tris-HCl (pH 8.0), 1 mM substrate dissolved in dimethyl sulfoxide (DMSO), 2 mM SAM, and 10 mg / mL purified SmOMT. After incubation at 37°C for 4 hours, the reaction was terminated by adding 20 mL of methanol, and the mixture was centrifuged at 12000 g for 20 minutes. Product purification was performed on a Shimadzu LC-20AT high-performance liquid chromatography system equipped with a reversed-phase COSMOSIL 5C18-MS-II column (250 mm × 10 mm, 5 μm). The structure of the purified product was characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

[0062] SmOMT methylated seven flavonoids (2, 3, 6, 8-11) with conversions ranging from 5% to 80%, and produced only monomethylated products, indicating its strict regioselectivity for flavonoids. To determine the methylation sites, products 2a (where compound 2a represents the methylated product of compound 2, and the other compounds are represented in the same way), 6a, 8a, 9a, and 10a were isolated and purified, and their structures were characterized by NMR. The results identified products 2a, 6a, 8a, and 9a as 3'-methoxyflavones, and product 10a as 3-methoxykaempferol. These results confirm that SmOMT tends to methylate the 3'-OH of flavonoids. SmOMT methylated all six tested coumarin compounds (12-17) with conversions ranging from 5% to 95%. Except for aesculin (13), which produced a dimethylated product, the other coumarin substrates produced only monomethylated derivatives. For heptanoside (13), three methylated products were detected: the major product 13a (which had the same retention time as isoscopolamine), a minor product corresponding to scopolamine (15), and a dimethylated derivative (product 13c). These results indicate that SmOMT has limited regioselectivity for coumarin compounds. Notably, methylation of 7-amino-4-methylcoumarin (17) was detected, confirming its weak N-methylation activity. SmOMT catalyzed the methylation of nine acetophenone derivatives (19, 21, 22, 24, 25, ...). Methylation of compounds 27, 28, 31, and 32) was performed with conversions ranging from 15% to 100%. Almost all substrates produced a single monomethylated derivative, indicating a strong regioselectivity for acetophenone compounds. Structural analysis of products 19a, 22a, 24a, 25a, 28b, 31a, and 32a by NMR and comparison with standards confirmed that methylation occurred at the meta-hydroxyl group (3'-OH / 5'-OH), demonstrating that SmOMT has a strong preference for the meta-hydroxyl group in acetophenone compounds. Compound 21, containing an amino group... N -Methylation further confirms that SmOMT has both... O - / N -Methylation ability, results as follows Figure 3-13 As shown.

[0063] Example 5: Mutant SmOMT M2 Construction and activity analysis

[0064] Using pET28a(+)-SmOMT as a template, site-directed mutagenesis was performed on SmOMT using mutagenic primers. The purified PCR product was then... Dpn The plasmid was digested with enzyme I, and the digestion product was then transformed into E. coli DH5α competent cells. The mutant plasmid was verified by DNA sequencing, and the verified plasmid was transformed into E. coli Rosetta (DE3) for heterologous protein expression.

[0065] Under the conditions of Example 4, the activities of the E152A and I306A single mutants were approximately 2.3-fold and 1.5-fold higher than those of the wild-type (WT), respectively. Combining these mutants yielded the double mutant SmOMT. E152A / I306A (The figure shows E152A / I306A), whose catalytic activity is 2.6 times higher than that of the wild type. Furthermore, compared to the wild type, the double mutant SmOMT... E152A / I306A It also exhibits stronger catalytic activity for other substrates, as shown in the following results. Figure 14 As shown.

[0066] Example 6: SmOMT-AtHMT Coupling Reaction and Construction of Fusion Enzyme

[0067] The method for constructing the pET28a(+)-AtHMT recombinant vector is as previously described. Using compound 31 as a substrate, SmOMT... M2 With AtHMT V140T The catalytic activity of the combination was evaluated. The reaction was carried out in a 100 μL mixture containing 50 mM Tris-HCl-150 mM NaCl buffer (pH 8.0) and 10 μg of purified SmOMT. M2 1 μg of purified AtHMT V140T The reaction mixture consisted of 0.8 mM substrate, 4 mM SAH, and 10 mM iodomethane (CH3I). After incubation overnight at 37 °C, 100 μL of methanol was added to terminate the reaction, followed by centrifugation at 12000 rpm for 20 min. The supernatant was analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The reaction progress was monitored by HPLC at seven time points over a period of 0.5 to 8 hours. AtHMT... V140T and SmOMT M2 Using a template, a fusion enzyme was constructed via PCR amplification using specific primers (with a connector introduced simultaneously). A fusion plasmid was constructed via multi-fragment homologous recombination and verified by DNA sequencing.

[0068] Upstream primer (F1):

[0069] GGTGCCGCGCGGCAGCCATATGATGGCGGAAGAACAGCAGAACTCTG

[0070] Downstream primer (FE-2-F2):

[0071] CACCGCCGACGCCGACCCCGGGGTCTGCCGGGGGAGTGATCGTGGA

[0072] Downstream primer (FE-2-R1):

[0073] GGGGTCGGCGTCGGCGGTGTCGGGGTCGGGTTGATCTTTTTCCAACGACCCAGTTTTT

[0074] Downstream primer (R2):

[0075] ctcgagtgcggccgcaagcttTCACTTGTGGAACTCCACTACTGAT.

[0076] Time-progression experiments showed that the methylation reaction of compound 31 proceeded stepwise, and the reaction was completed after 8 hours, as shown in the results. Figure 15 As shown. Simultaneously, various connector systems were compared with free enzyme cascade systems. Figure 15 In this context, NFE stands for AtHMT (Free Enzyme Cascade System). V140T and SmOMT M2 (All exist and are not connected), FE-1, FE-2, FE-3 and FE-4 are AtHMT V140T and SmOMT M2 Enzyme cascade systems that connect via different common connectors, wherein FE-2 is an enzyme cascade system connected via L... 95 Establish a connection. From Figure 15 The results show that FE-2 has better catalytic activity than NFE.

[0077] Example 7: Optimization of fusion enzyme reaction system and analysis of anti-inflammatory activity of methylated products

[0078] 1. Using luteolin (2) as a substrate, the reaction system catalyzed by the fusion enzyme and independent of S-adenosylmethionine (SAM) was optimized. Four key parameters were investigated: the molar ratio of iodomethane to S-adenosylhomocysteine ​​(tested ratios were 5:1, 4:1, 3:1, 2:1, 1:1, and 1:2), reaction time (5–420 min), pH (3.0–11.0), and temperature (4–60 °C). The reaction was terminated before HPLC analysis, and all optimization experiments were performed in triplicate.

[0079] 2. RAW264.7 cells were incubated with substrates or methylated products (concentrations of 2.5–60 μM) at 37°C for 48 hours. Experimental groups included lipopolysaccharide (LPS) treatment (1.2 μg / mL) and an untreated control group. The culture supernatant was then discarded. The accumulation of nitric oxide (NO) in the cell-free supernatant was measured using the Griess reaction to assess the activity of inducible nitric oxide synthase (iNOS).

[0080] After optimization of the FE-2 system, the highest conversion rate was achieved under the following conditions: 37℃, pH 6.0 (50mM Na2HPO4–NaH2PO4 buffer), CH3I / SAH molar ratio of 2, and reaction time of 3 hours. To evaluate the potential of the optimized system, scale-up experiments were conducted. These experiments successfully prepared products 2a, 8a, 12a, and 31a without the use of expensive SAM, as shown in the results. Figure 16 As shown in the figure. Anti-inflammatory experiments indicated that the methylated product exhibited a stronger inhibitory effect on NO compared to its parent compound. Notably, product 8a showed significantly enhanced activity, with its half-maximal inhibitory concentration (IC50) being significantly higher. 50 The concentration was 4.38 μM, compared to compound 8 (IC). 50 =9.39μM) decreased by 50%, as shown in the following results Figure 17 As shown.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An O-methyltransferase mutant SmOMT M2 Its characteristics are, It was obtained by double mutation of E152A and I306A based on the O-methyltransferase SmOMT with the amino acid sequence SEQ ID NO.

1.

2. A fusion enzyme, characterized in that, SmOMT as described in claim 1 M2 With mutant halide methyltransferase AtHMT V140T Through L 95 It is formed by linking together, and its amino acid sequence is SEQ ID NO.

2.

3. A SmOMT M2 The encoding gene, characterized in that, For encoding SmOMT as described in claim 1 M2 .

4. A recombinant vector, characterized in that, It contains the gene as described in claim 3.

5. A recombinant host cell, characterized in that, It contains the recombinant vector as described in claim 4 or carries the gene as described in claim 3.

6. The O-methyltransferase mutant SmOMT as described in claim 1 M2 Alternatively, the application of the fusion enzyme as described in claim 2 in catalyzing methylation reactions, wherein the substrate for the methylation reaction is selected from caffeic acid, luteolin, geraniol, sennaol, quercetin, isorhamnetin, kaempferol, sennain, xanthophyllol, fraxin, scopolamine, isohyoscyamine, umbelliferone, 7-aminocoumarin, 3'-hydroxyacetophenone, 3'-aminoacetophenone, 3'-hydroxy-4'-methoxyacetophenone, 4'-hydroxy-3'-methoxyacetophenone, 2',3'-dihydroxyacetophenone, 2',5'-dihydroxyacetophenone, 3',5'-dihydroxyacetophenone, 2',3',4'-trihydroxyacetophenone, 2',3'-dihydroxy-4'-methoxyacetophenone, resveratrol, and 1,3,8-trihydroxyanthraquinone; and SAM is used as the methyl donor in the methylation process.

7. The application of the fusion enzyme as described in claim 2 in SAM-independent methylation, wherein the methylation process is as follows: CH3I and SAH are used as methyl donors.

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