Three-dimensional crystal structure and application of PsiM protein, SAH and Norbaeocystin compound of stropharia rugoso-annulata

Abstract

The invention provides a three-dimensional crystal structure of a PsiM protein, SAH and Norbaeocystin compound, and belongs to the technical field of biology, the compound comprises the PsiM protein, SAH and Norbaeocystin, the PsiM protein has an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence with at least 80% of sequence identity with the amino acid sequence as shown in SEQ ID NO.1, and the PsiM protein has an amino acid sequence as shown in SEQ ID NO.2 or an amino acid sequence with at least 80% of sequence identity with the amino acid sequence as shown in SEQ ID NO.2 or an amino acid sequence with at least 80% of sequence identity with the amino acid sequence as shown in SEQ ID NO.2. The atoms in the three-dimensional crystal structure of the compound have at least 80% of atomic coordinates listed in Table 1; or the square root variance of the atomic structure coordinates of the main chain carbon skeleton of at least 30% of amino acids and the atomic coordinates in the table 1 is smaller than or equal to # imgabs0 # imgabs1 #. The three-dimensional crystal structure is beneficial to clarification of a methyl transfer mechanism of PsiM protein in the Psilocybin biosynthesis process, so that structural basic guidance is better provided for Psilocybin biosynthesis; and the wide clinical application of the intermediate product Norbaeocystin in the traditional Chinese medicine is explored in an auxiliary manner. The invention further provides application of the three-dimensional crystal structure of the PsiM protein, the SAH and the Norbaeocystin compound of the stropharia rugoso-annulata to guidance and improvement of the biosynthesis yield of the Psilocybin.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a three-dimensional crystal structure and application of a Psilocybin mushroom PsiM protein, SAH and Norbaeocystin complex. Background Art

[0002] Psilocybin is a tryptamine hallucinogen. It was isolated from Psilocybe mexicana by Albert Hofmann in 1958 and is the first category of psychotropic drugs regulated by my country. Psilocybin is a neurotoxin with neuropsychedelic effects. Its structure belongs to the indole alkaloids and is a tryptamine derivative named 4-phospho-N,N-dimethyltryptamine. At present, most of the research on psilocybin focuses on the treatment of moderate to severe and refractory depression. Its advantages in treating depression are: fast onset and long-lasting effect, lower addiction than other hallucinogens, and fewer side effects. As a new antidepressant, psilocybin provides new hope for patients with moderate to severe and refractory depression. However, there are still great obstacles to its application in clinical practice as a hallucinogen. In the future, a lot of scientific research is needed to clarify its safety, feasibility and effectiveness.

[0003] The Psilocybin methyltransferase PsiM protein belongs to the methyltransferase family and plays a role in methyl transfer during the biosynthesis of Psilocybin. Under the mediation of its cofactor SAM (S-adenosylmethionine), the Psilocybin PsiM protein transfers a methyl group to the tail amino group of Norbaeocystin to generate the Baeocystin substrate, and then catalyzes the addition of a new methyl group to the tail amino group of Baeocystin to generate Psilocybin. The analysis of the three-dimensional crystal structure of the PsiM methyltransferase protein complex helps to clarify the mechanism of methyl transfer during the biosynthesis of Psilocybin and the clinical role of the intermediate product Norbaeocystin in the synthetic route, thereby providing a better structural basis for the biosynthesis of Psilocybin and exploring the wider clinical application of its intermediate product Norbaeocystin. Summary of the invention

[0004] The purpose of the present invention is to provide a three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin, which is helpful to clarify the mechanism of methyl transfer of Psilocybin PsiM protein in the process of Psilocybin biosynthesis, so as to better provide structural basis guidance for Psilocybin biosynthesis and assist in exploring the wider clinical application of its intermediate product Norbaeocystin.

[0005] The present invention also provides the application of the three-dimensional crystal structure of the Psilocybin PsiM protein, SAH and Norbaeocystin complex in guiding the improvement of the Psilocybin biosynthesis yield.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a three-dimensional crystal structure of a complex of Psilocybin PsiM protein, SAH (S-adenosylhomocysteine) and Norbaeocystin, wherein the complex comprises Psilocybin PsiM protein, SAH and Norbaeocystin, wherein the Psilocybin PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto;

[0008] The atoms in the three-dimensional crystal structure of the complex have at least 80% of the atomic coordinates listed in Table 1, or the square root variance of the atomic structure coordinates of the main chain carbon skeleton of at least 30% of the amino acids and the atomic coordinates in Table 1 is less than or equal to

[0009] Furthermore, the resolution of the three-dimensional crystal structure of the complex reaches

[0010] The space group of the three-dimensional crystal structure of the complex is P2 1 2 1 2;

[0011] The unit cell parameters of the three-dimensional crystal structure of the complex are: α=β=γ=90°.

[0012] Furthermore, the three-dimensional crystal structure of the complex contains one Psilocybin PsiM protein, one SAH molecule and one Norbaeocystin molecule in one crystallographic asymmetric unit, and the secondary structure of the Psilocybin PsiM protein in the three-dimensional crystal structure of the complex includes 12 α-helices and 9 β-folds;

[0013] Among them, α helix 1, that is, the amino acid segment containing Tyr13-Ala19; α helix 2, that is, the amino acid segment containing Pro21-Lys24; β fold 1, that is, the amino acid segment containing Val27-Ans30; β fold 2, that is, the amino acid segment containing Gly33-Val37; α helix 3, that is, the amino acid segment containing Pro42-Phe57; α helix 4, that is, the amino acid segment containing Val72-Leu93; β fold 3, that is, the amino acid segment containing Ile101-Ile106; α helix 5, that is, the amino acid segment containing Ile113-Arg121; β fold 4, that is, the amino acid segment containing Trp125-Glu131; α helix 6, that is, the amino acid segment containing Arg134-Asn147; β fold 5, that is, the amino acid segment containing Leu153-Glu15 7; α helix 7, i.e. the amino acid segment containing Val166-Glu170; β fold 6, i.e. the amino acid segment containing Phe179-Met181; α helix 8, i.e. the amino acid segment containing Ala190-Thr195; α helix 9, i.e. the amino acid segment containing Val214-Met217; α helix 10, i.e. the amino acid segment containing Ala225-Leu237; β fold 7, i.e. the amino acid segment containing Trp243-Leu248; α helix 11, i.e. the amino acid segment containing Leu251-Leu264; β fold 8, i.e. the amino acid segment containing Thr269-GLu276; β fold 9, i.e. the amino acid segment containing Thr279-Trp286; α helix 12, i.e. the amino acid segment containing Pro303-Ser306.

[0014] Furthermore, in the three-dimensional crystal structure of the complex, the side chain of Thr158 hydrogen bonds with the N1 position of the SAH adenine base, the side chain of Glu131 hydrogen bonds with the ribose, the side chain of Arg75 and the side chain of Asn183 hydrogen bonds with the SAH carboxyl group, and the main chain of Thr108 provides a hydrogen bond for the SAH molecule; the side chains of Arg75 and Arg281, as well as the side chain of Asn183 are directly hydrogen bonded to the phosphate group of Norbaocystin, and are also hydrogen bonded to the main chain of Gly203; the main chain of Cys69 and the side chain of Asn74 also form hydrogen bonds with the phosphate group through water molecules, and the hydroxyl group of Asn183 and the main chain of Pro184 form hydrogen bonds with the amine group of the indole ring of Norbaeocystin.

[0015] Based on the same inventive concept, the present invention provides a method for expressing and purifying Psilocybin mushroom PsiM protein, the expression and purification method comprising:

[0016] A fusion plasmid of Psilocybin PsiM gene and pET28a-Sumo was constructed and transformed into Escherichia coli to express Psilocybin PsiM protein containing a specific tag;

[0017] The Psilocybe occidentalis PsiM protein containing the specific tag is separated by a method that specifically recognizes the specific tag, the Psilocybe occidentalis PsiM protein is separated by Ni affinity chromatography after the specific tag is cleaved, and then a high-purity Psilocybe occidentalis PsiM protein is obtained by gel filtration chromatography;

[0018] Wherein, the Escherichia coli includes Escherichia coli BL21 (DE3);

[0019] The Psilocybe occidentalis PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto.

[0020] Furthermore, the construction of the fusion plasmid of the Psilocybe PsiM gene and pET28a-Sumo, transformation into Escherichia coli, and expression of the Psilocybe PsiM protein containing a specific tag specifically includes:

[0021] The Psilocybe PsiM gene was cloned into the pET28a-Sumo plasmid, and the N-terminus of the Psilocybe PsiM gene was tagged with a His6-SUMO tag to obtain a fusion plasmid;

[0022] The fusion plasmid was transformed into Escherichia coli to express the Psilocybin mushroom PsiM protein containing the His6-SUMO tag.

[0023] Based on the same inventive concept, the present invention provides a method for crystallizing a complex of Psilocybin PsiM protein, SAH and Norbaeocystin, the crystallization method comprising:

[0024] The Psilocybin PsiM protein solution was concentrated to 10 mg / ml, then dissolved by adding SAH and Norbaeocystin, and incubated on ice for 60 min to obtain a mixed solution;

[0025] Crystallization reagents were used as initial screening conditions for crystal growth, and sitting drop or hanging drop vapor diffusion methods were used for crystallization at a crystallization temperature of 18°C ​​to obtain crystals of Psilocybin PsiM protein, SAH, and Norbaeocystin complex;

[0026] Wherein, the Psilocybin PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto;

[0027] In the mixed solution, the molar ratio of the Psilocybin PsiM protein, the SAH and the Norbaeocystin is 1:4:10.

[0028] Furthermore, the volume ratio of the mixed solution to the crystallization reagent is 3:1 to 1:3;

[0029] In the crystallization using the sitting drop or hanging drop vapor diffusion method, the crystallization conditions are 40% (v / v) isopropanol, 0.1 M imidazole pH 6.5, and 15% (w / v) polyethylene glycol 8000.

[0030] Based on the same inventive concept, the present invention provides the application of the three-dimensional crystal structure of the Psilocybin PsiM protein, SAH and Norbaeocystin complex in guiding the improvement of Psilocybin biosynthesis yield.

[0031] Based on the same inventive concept, the present invention provides the application of the three-dimensional crystal structure of the Psilocybin mushroom PsiM protein, SAH and Norbaeocystin complex in assisting the screening of antidepressant or antianxiety drugs.

[0032] Based on the same inventive concept, the present invention provides the application of the three-dimensional crystal structure of the Psilocybin PsiM protein, SAH and Norbaeocystin complex in assisting the regulation of the methyl transfer step in the Psilocybin biosynthesis process.

[0033] Based on the same inventive concept, the present invention also provides the use of Norbaeocystin as or in the preparation of antidepressant or antianxiety drugs.

[0034] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0035] The three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin of the present invention, wherein the complex comprises Psilocybin PsiM protein, SAH and Norbaeocystin, wherein the Psilocybin PsiM protein contains 309 amino acids and has a molecular weight of 34.3 kDa, and the three-dimensional crystal structure of the complex of the Psilocybin PsiM protein, the cofactor SAH and the substrate Norbaeocystin is obtained by X-ray diffraction with a resolution of Its space group is P2 1 2 12. The secondary structure of Psilocybin PsiM protein in the crystal structure of the complex is composed of 12 α-helices and 9 β-folds. As an important methyltransferase in the biosynthesis of psilocybin, Psilocybin PsiM protein transfers methyl to the tail amino group of Norbaeocystin under the action of its cofactor SAM (S-adenosylmethionine) to generate Baeocystin substrate, and then catalyzes the addition of new methyl to the tail amino group of Baeocystin to generate Psilocybin. Therefore, analyzing the three-dimensional crystal structure of the complex of Psilocybin PsiM protein with cofactor SAH and substrate Norbaeocystin will help to clarify the mechanism of methyl transfer in the biosynthesis of Psilocybin, as well as the clinical role of the intermediate product Norbaeocystin in the synthetic route, thereby providing a better structural basis for the biosynthesis of Psilocybin and exploring the wider clinical application of its intermediate product Norbaeocystin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 These are the results of gel filtration chromatography and SDS-PAGE detection of Psilocybin PsiM protein.

[0038] Figure 2 This is a crystal image of the complex between Psilocybin PsiM protein, cofactor SAH and substrate Norbaeocystin.

[0039] Figure 3 Schematic diagram of the three-dimensional structure of the complex of Psilocybin PsiM protein, cofactor SAH and substrate Norbaeocystin: (A) Cartoon diagram of the three-dimensional structure of the complex of Psilocybin PsiM protein, cofactor SAH and substrate Norbaeocystin; (B) Surface charge distribution diagram of the complex of Psilocybin cofactor SAH and substrate Norbaeocystin.

[0040] Figure 4 This is the thin layer chromatography (TLC) result of the reaction between the Psilocybin PsiM protein mutant and the cofactor SAM catalyzed substrate Norbaeocystin.

[0041] Figure 5The efficacy of Psilocybin and its synthetic intermediates Norbaeocystin and Baeocystin on depression in animal experiments.

[0042] Figure 6 This is the plasmid map of the recombinant vector. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0044] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0045] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0046] The three-dimensional crystal structure and application of the Psilocybin PsiM protein, SAH and Norbaeocystin complex of the present application will be described in detail below in combination with the examples and experimental data.

[0047] Example 1

[0048] Expression and purification of the full-length protein of Psilocybin methyltransferase PsiM, including:

[0049] 1) The Psilocybin PsiM Met1-Phe309 gene was cloned into the pET28a-Sumo vector by molecular cloning technology to express the fusion protein with His6-SUMO tag fused to the nitrogen end, and the recombinant vector was obtained (see the vector plasmid map for details). Figure 6 ).

[0050] The nucleotide sequence of the full-length gene of Psilocybin mushroom PsiM is shown in SEQ ID NO. 2. The upstream primer used for PCR amplification of the Psilocybin mushroom PsiM gene is psiM-BamHI-1F: 5'-CACAGGATCCATGCATATCAGAAATCCTTACCGTACA-3', and the downstream primer is psiM-XhoI-309R: 5'-GCACCTCGAGCTAGAAAAGAGAGCTGAGCTCGGG-3'.

[0051] 2) Transform the recombinant vector into the E. coli BL21 (DE3) strain (PsiM protein is highly soluble and expressed in the E. coli BL21 (DE3) expression strain, and the purity and uniformity are good). Add kanamycin sulfate to 1L LB liquid medium to make the final concentration 50ug / ml, and continue to shake culture at 37°C and 220rpm until the OD reaches 0.6-0.8 (about 3h), then add 200ul 1M IPTG to each liter of culture to make the final IPTG concentration 0.2mM, and finally induce overnight at 18°C ​​and 220rpm for 16-20h. Next, collect the induced expression bacterial solution in a bacterial collection bucket, centrifuge at 6000rpm and 4°C for 10min, discard the supernatant, precipitate the bacteria, and resuspend and wash the bacteria with 20mL 1x PBS. The bacterial resuspension was transferred to a 50 ml centrifuge tube, centrifuged at 4000 rpm and 4°C for 15 min, the supernatant was discarded, and the bacterial cells were harvested for protein purification.

[0052] 3) Add 5 times the volume of the bacteria (the bacteria concentration is easiest to break between 10% and 20%) of the equilibrium buffer (containing 20mM Tris pH 8.0, 500mM NaCl, 25mM imidazole), and fully blow to resuspend the bacteria. Precool, clean, and rinse the high-pressure crusher with the equilibrium buffer, and repeatedly break the bacteria at 1200-1500bar for 3-4 times (after breaking, it should flow out like water and not be viscous). Transfer the broken bacteria solution to a high-speed centrifuge tube and centrifuge at 17,000rpm and 4℃ for 1 hour. After centrifugation, discard the precipitate and transfer the supernatant to a 50ml centrifuge tube.

[0053] 4) Separate the target protein by Ni affinity chromatography. The separation principle is: low imidazole concentration buffer for loading and high imidazole concentration buffer for elution. The specific operation is as follows:

[0054] The supernatant of high-speed centrifugation was loaded onto a cleaned and equilibrated nickel column (5mL HisTrap pre-packed column) at a flow rate of 3ml / min. After loading, it was rinsed with 100ml of equilibration buffer and placed in an AKTA system for elution. The flow rate was 3ml / min and the gradient was set to "10min, 100% elution buffer (containing 20mM Tris pH 8.0, 500mM NaCl, 500mM imidazole)". When the A280 value began to increase, the protein was collected and the purification results were checked by SDS-PAGE. The eluted protein was added with ULP1 enzyme (100ul) and placed in a 3.5kDa dialysis bag, placed in 500ml of dialysis solution (containing 20mM Tris8.0, 500mMNaCl,) and dialyzed at 4℃ for 3 hours. The collected protein solution was loaded onto two series-connected cleaned and equilibrated nickel columns at a speed of 1ml / min. Use Bradford to detect the flow-through liquid. When Bradford starts to change color, start collecting the flow-through liquid immediately. After the protein sample is loaded, add the equilibration buffer and continue to pass through the column until Bradford detects that there is very little protein in the flow-through liquid.

[0055] 5) Next, the collected protein was concentrated using a 10 kDa cutoff concentrator, and the concentrated protein was further purified by gel filtration chromatography (Superdex 20016 / 600. Superdex, USA), and the purification results were checked by SDS-PAGE. The buffer used was 10 mM Tris pH 8.0, 100 mM NaCl. Figure 1 (Left) is the peak diagram of gel filtration chromatography. Figure 1 (Right) After gel filtration chromatography, the protein at the peak position was collected for gel electrophoresis and stained with Coomassie Brilliant Blue. Finally, the obtained protein was concentrated and stored at -80°C for crystallization experiments.

[0056] The present invention preferably uses the prokaryotic cell expression system of Escherichia coli (but does not exclude other expression systems, such as expression in other prokaryotes or other eukaryotic cells), expresses the above-mentioned protein in the form of a fusion protein, and preferably selects the His tag to obtain soluble expression (but does not exclude other tags, such as MBP or GST tags also have soluble expression).

[0057] Example 2

[0058] Crystallization experiment of the complex of Psilocybin PsiM protein with cofactor SAH and substrate Norbaeocystin.

[0059] 1) A PsiM protein concentration of 10 mg / ml is preferably selected for crystal growth, and the PsiM protein of this concentration is mixed with the cofactor SAH and the substrate Norbaeocystin in a molar ratio of 1:4:10, and incubated on ice for 60 minutes to obtain a mixed solution.

[0060] 2) Use the crystallization reagent in the crystallization kit (Index, Crystal Screen I / II from Hampton Research and other companies, and JCSG Core I-IV Suite from Qiagen, etc.) as the initial screening conditions for crystal growth. Perform hanging drop optimization on the initial screening conditions for the screened crystals, and use the hanging drop or sitting drop vapor diffusion method for crystallization. The volume ratio of the mixed solution to the crystallization reagent in the crystallization droplet is between 3:1 and 1:3. In this embodiment, the volume ratio is 1:1, and the crystallization temperature is 18°C.

[0061] The present invention obtains initial crystals under multiple different crystallization reagent conditions. Through subsequent optimization and adjustment, 40% (v / v) isopropanol, 0.1M imidazole pH 6.5, 15% (w / v) PEG 8000 are preferably used as crystal growth conditions. The obtained crystals are as follows Figure 2 As shown, a set of resolution X-ray diffraction data.

[0062] Specifically, the three-dimensional crystal structure of the complex of the Psilocybin methyltransferase PsiM protein, the cofactor SAH and the substrate Norbaeocystin has a PsiM protein molecule, a SAH molecule and a Norbaeocystin molecule in one crystallographic asymmetric unit, and its secondary structure consists of 12 α helices and 9 β folds, such as Figure 3 As shown in A.

[0063] Among them, α helix 1, that is, the amino acid segment containing Tyr13-Ala19; α helix 2, that is, the amino acid segment containing Pro21-Lys24; β fold 1, that is, the amino acid segment containing Val27-Ans30; β fold 2, that is, the amino acid segment containing Gly33-Val37; α helix 3, that is, the amino acid segment containing Pro42-Phe57; α helix 4, that is, the amino acid segment containing Val72-Leu93; β fold 3, that is, the amino acid segment containing Ile101-Ile106; α helix 5, that is, the amino acid segment containing Ile113-Arg121; β fold 4, that is, the amino acid segment containing Trp125-Glu131; α helix 6, that is, the amino acid segment containing Arg134-Asn147; β fold 5, that is, the amino acid segment containing Leu153-Glu15 7; α helix 7, i.e. the amino acid segment containing Val166-Glu170; β fold 6, i.e. the amino acid segment containing Phe179-Met181; α helix 8, i.e. the amino acid segment containing Ala190-Thr195; α helix 9, i.e. the amino acid segment containing Val214-Met217; α helix 10, i.e. the amino acid segment containing Ala225-Leu237; β fold 7, i.e. the amino acid segment containing Trp243-Leu248; α helix 11, i.e. the amino acid segment containing Leu251-Leu264; β fold 8, i.e. the amino acid segment containing Thr269-GLu276; β fold 9, i.e. the amino acid segment containing Thr279-Trp286; α helix 12, i.e. the amino acid segment containing Pro303-Ser306.

[0064] Among them, the side chain of Thr158 hydrogen bonds with the N1 position of the SAH adenine base, the side chain of Glu131 hydrogen bonds with the ribose, the side chains of Arg75 and Asn183 hydrogen bond with the SAH carboxyl group, and the main chain of Thr108 provides a hydrogen bond for the SAH molecule; the side chains of Arg75 and Arg281, as well as the side chain of Asn183 are directly hydrogen bonded to the phosphate group of norbaocystin, and are also hydrogen bonded to the main chain of Gly203; the main chain of Cys69 and the side chain of Asn74 also form hydrogen bonds with the phosphate group through water molecules. In addition, the hydroxyl group of Asn183 and the main chain of Pro184 form hydrogen bonds with the amine group of the indole ring of Norbaeocystin.

[0065] From the surface charge distribution of the PsiM protein and SAH complex, it can be seen that both the cofactor SAH and the substrate binding pocket are surrounded by negative charges. Figure 3 B).

[0066] This three-dimensional crystal structure describes the secondary structure composition of the PsiM protein, the direction of the peptide chain, the organizational pattern, the three-dimensional molecular structure, its binding mode with SAH and Norbaeocystin, and the methyl transfer mechanism.

[0067] The atomic coordinate groups of the crystal three-dimensional structure of the complex of Psilocybin methyltransferase PsiM protein, cofactor SAH and substrate Norbaeocystin are as follows:

[0068] Crystal space group: P2 1 2 1 2;

[0069] The unit cell parameters are: α=β=γ=90°;

[0070] I / σI: 10.9 (4.8);

[0071] Rwork and Rfree are: 0.1759 / 0.2173;

[0072] Resolution range 50.00-2.50.

[0073] Data completeness (%): 99.9 (100.0).

[0074] The atomic coordinates are shown in Table 1:

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Data collection and correction statistics

[0120]

[0121] *Highest resolution included in brackets.

[0122] Example 3

[0123] 1. To verify the methyl transfer effect of PsiM of Psilocybin mushroom, based on the analysis of the three-dimensional crystal structure of the PsiM-SAH-Norbeaocystin complex, the key amino acids for substrate binding and catalysis were mutated. Overlap PCR was used to mutate L68, N74, R75, E131, N183, F186, Y187, F202, H210, M217, F226, and R281 into alanine, and P184 and P185 into glycine. Subsequently, 200uM mutant PsiM protein was incubated with 2.5mM SAM and 2.5mM Norbeaocystin at 25℃ for 1h for thin layer chromatography experiments (20ml of developing agent was poured into one side of the glass cylinder, and the chromatographic silica gel plate was pre-saturated in the cylinder for 1h after being spotted with a capillary tube, and then developed for 3h) to verify its catalytic activity. The results are as follows Figure 4As shown in the figure, TLC results show that PsiM can catalyze the conversion of Norbaocystin to Baeocystin. Mutations of residues that interact hydrophobically with the substrate have a greater impact on catalytic ability than mutations of residues that interact hydrophilically with the substrate. For example, L68A, Y187A, F202A, M217A, and F226A showed a significant decrease or disappearance in the catalytic ability of Norbaeocystin substrate, while H210A had little effect on the catalytic efficiency of PsiM. In contrast to these hydrophobic contacts, mutations of hydrophilic residues, such as R74A and R75A, also slightly reduced the catalytic activity of PsiM, but R281A lost the catalytic ability for the substrate. The other five mutants (including E131A and "NPPF" (N183A, P184G, P185G, and F186A) are mutants required for the binding of the auxiliary factor SAM) all showed decreased catalytic ability or loss of methyltransferase activity. In the "NPPF" motif, N183A had a relatively small effect on the enzyme activity of PsiM.

[0124] 2. To test the efficacy of intermediates in the synthesis pathway of Psilocybin in anti-depression and anxiolysis, a depression model of C57BL / 6J mice was first established (mice received 100 foot shocks (0.45 mA, 1 s, randomly spaced 1 h on the 1st and 4th days; tail suspension stress was performed on the 2nd and 5th days; mice were confined in a tube for 1 h on the 3rd and 6th days). Next, Psilocybin, Beaocystin, and Norbaeocystin were dissolved in 0.9% sterile saline solution and injected intraperitoneally (1 mg / kg) on ​​the morning of the first day after modeling. Blank control: The control group was injected with 0.9% sterile saline, and behavioral tests (Splash test, Social interaction test, Novelty-suppressedfeeding test, Open field test, Forced swim test, and Sucrose preference) were performed on the second day after injection. Test (sugar water preference test), followed by brain tissue sectioning (refer to "The Mouse Brain in Stereotaxic Coordinates" by Paxinos and Franklin to select the section area), and image analysis can be performed after immunofluorescence staining.

[0125] like Figure 5As shown, the c-FOS factor (a marker of neuronal activity) activity in the prefrontal cortex (PFC) and midbrain ventral tegmentum (VTA) regions was Norbaeocystin>Psilocybin>Beaocystin>blank control; the c-FOS factor activity in the striatum (NAC) region was Psilocybin>Norbeaocystin>Beaocystin>blank control. The results show that Norbaeocystin, an intermediate product of the Psilocybin synthesis pathway of Psilocybin, is better than Psilocybin in terms of antidepressant efficacy and reduced side effects.

[0126] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0127] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0128] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin, characterized by: The complex comprises a Psilocybin PsiM protein, SAH and Norbaeocystin, wherein the Psilocybin PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto; The atoms in the three-dimensional crystal structure of the complex have at least 80% of the atomic coordinates listed in Table 1, or the square root variance of the atomic structure coordinates of the main chain carbon skeleton of at least 30% of the amino acids and the atomic coordinates in Table 1 is less than or equal to 2. The three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to claim 1, characterized in that: The resolution of the three-dimensional crystal structure of the complex reaches The space group of the three-dimensional crystal structure of the complex is P21212; The unit cell parameters of the three-dimensional crystal structure of the complex are: α=β=γ=90°.

3. The three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to claim 1, characterized in that: The three-dimensional crystal structure of the complex contains one Psilocybin PsiM protein, one SAH molecule and one Norbaeocystin molecule in one crystallographic asymmetric unit, and the secondary structure of the Psilocybin PsiM protein in the three-dimensional crystal structure of the complex includes 12 α-helices and 9 β-folds; Among them, α helix 1, that is, the amino acid segment containing Tyr13-Ala19; α helix 2, that is, the amino acid segment containing Pro21-Lys24; β fold 1, that is, the amino acid segment containing Val27-Ans30; β fold 2, that is, the amino acid segment containing Gly33-Val37; α helix 3, that is, the amino acid segment containing Pro42-Phe57; α helix 4, that is, the amino acid segment containing Val 72-Leu93; β fold 3, that is, the amino acid segment containing Ile101-Ile106; α helix 5, that is, the amino acid segment containing Ile113-Arg121; β fold 4, that is, the amino acid segment containing Trp125-Glu131; α helix 6, that is, the amino acid segment containing Arg134-Asn147; β fold 5, that is, the amino acid segment containing Leu153-Glu157; α helix 7, that is, the amino acid segment containing Val 166-Glu170 amino acid segment; β fold 6, i.e., the amino acid segment containing Phe179-Met181; α helix 8, i.e., the amino acid segment containing Ala190-Thr195; α helix 9, i.e., the amino acid segment containing Val214-Met217; α helix 10, i.e., the amino acid segment containing Ala225-Leu237; β fold 7, i.e., the amino acid segment containing Trp243-Leu248; α helix 11, i.e., the amino acid segment containing Leu251-Leu264; β fold 8, i.e., the amino acid segment containing Thr269-GLu276; β fold 9, i.e., the amino acid segment containing Thr279-Trp286; α helix 12, i.e., the amino acid segment containing Pro303-Ser306.

4. The three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to claim 1, characterized in that: In the three-dimensional crystal structure of the complex, the side chain of Thr158 hydrogen bonds with the N1 position of the SAH adenine base, the side chain of Glu131 hydrogen bonds with the ribose, the side chains of Arg75 and Asn183 hydrogen bond with the SAH carboxyl group, and the main chain of Thr108 provides a hydrogen bond for the SAH molecule; the side chains of Arg75 and Arg281, as well as the side chain of Asn183, are directly hydrogen bonded to the phosphate group of Norbaocystin, and are also hydrogen bonded to the main chain of Gly203; the main chain of Cys69 and the side chain of Asn74 also form hydrogen bonds with the phosphate group through water molecules, and the hydroxyl group of Asn183 and the main chain of Pro184 form hydrogen bonds with the amine group of the indole ring of Norbaeocystin.

5. A method for expressing and purifying Psilocybin PsiM protein, characterized in that: The expression and purification method comprises: A fusion plasmid of Psilocybin PsiM gene and pET28a-Sumo was constructed and transformed into Escherichia coli to express Psilocybin PsiM protein containing a specific tag; The Psilocybe occidentalis PsiM protein containing the specific tag is separated by a method that specifically recognizes the specific tag, the Psilocybe occidentalis PsiM protein is separated by Ni affinity chromatography after the specific tag is cleaved, and then a high-purity Psilocybe occidentalis PsiM protein is obtained by gel filtration chromatography; Wherein, the Escherichia coli includes Escherichia coli BL21 (DE3); The Psilocybe occidentalis PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto.

6. The method for expressing and purifying Psilocybin mushroom PsiM protein according to claim 5, characterized in that: The construction of the fusion plasmid of Psilocybin PsiM gene and pET28a-Sumo, transformation into Escherichia coli, and expression of Psilocybin PsiM protein containing a specific tag specifically includes: The Psilocybe PsiM gene was cloned into the pET28a-Sumo plasmid, and the N-terminus of the Psilocybe PsiM gene was tagged with a His6-SUMO tag to obtain a fusion plasmid; The fusion plasmid was transformed into Escherichia coli to express the Psilocybin mushroom PsiM protein containing the His6-SUMO tag.

7. A method for crystallizing a complex of Psilocybin PsiM protein, SAH and Norbaeocystin, characterized in that: The crystallization method comprises: The Psilocybin PsiM protein solution was concentrated to 10 mg / ml, then dissolved by adding SAH and Norbaeocystin, and incubated on ice for 60 min to obtain a mixed solution; Crystallization reagents were used as initial screening conditions for crystal growth, and sitting drop or hanging drop vapor diffusion methods were used for crystallization at a crystallization temperature of 18°C ​​to obtain crystals of Psilocybin PsiM protein, SAH, and Norbaeocystin complex; Wherein, the Psilocybin PsiM protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto; In the mixed solution, the molar ratio of the Psilocybin PsiM protein, the SAH and the Norbaeocystin is 1:4:

10.

8. The method for crystallizing the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to claim 7, characterized in that: The volume ratio of the mixed solution to the crystallization reagent is 3:1 to 1:3; The crystallization is carried out by sitting drop or hanging drop vapor diffusion method, and the crystallization conditions are 40% (v / v) isopropanol, 0.1M imidazole pH 6.5, and 15% (w / v) polyethylene glycol 8000.

9. Use of the three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to any one of claims 1 to 4 in guiding the improvement of Psilocybin biosynthesis yield.

10. Use of the three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to any one of claims 1 to 4 in assisting the screening of antidepressant or antianxiety drugs.

11. Use of the three-dimensional crystal structure of the complex of Psilocybin PsiM protein, SAH and Norbaeocystin according to any one of claims 1 to 4 in assisting the regulation of the methyl transfer step in the biosynthesis process of Psilocybin.

12. Use of Norbaeocystin as or in the preparation of an antidepressant or antianxiety drug.

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