Three-dimensional crystal structure of PsiD protein truncated body and tryptamine compound, crystallization method and application

Abstract

The invention provides a three-dimensional crystal structure of a compound of a PsiD protein truncation and tryptamine, and belongs to the technical field of biology, the compound comprises the PsiD protein truncation and the tryptamine, the PsiD protein truncation has an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence with at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO.1; 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 #. The three-dimensional crystal structure is beneficial to clarification of the decarboxylation mechanism of the PsiD protein of stropharia rugoso-annulata in the Psilocybin biosynthesis process and the self-inhibition mechanism of the Psilocybin on the W27 site at the N end, so that biotechnical guidance is better provided for Psilocybin biosynthesis, and the application of the stropharia rugoso-annulata PsiD protein in the biosynthesis process of stropharia rugoso-annulata PsiD protein in the biosynthesis process is facilitated. And meanwhile, more possibilities are provided for improving the yield of the Psilocybin. The invention further provides a crystallization method and application of the PsiD protein truncation and tryptamine compound of the pleurotus nummularia.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a three-dimensional crystal structure, a crystallization method and an application of a complex of a truncated Psilocybin PsiD protein and tryptamine. Background Art

[0003] Psilocybin belongs to the tryptamine class of hallucinogens and is a neurotoxin with neuropsychedelic effects. Its structure belongs to the indole alkaloid class. Some studies classify it as a non-selective 5-hydroxytryptamine 2A receptor agonist. Psilocybin naturally exists in mushrooms of the genus Psilocybin and is distributed all over the world. A large number of studies have shown that psilocybin, as a hallucinogen, has relatively low addictiveness and fewer adverse reactions. Its combination with psychotherapy has a significant effect in the treatment of depression, anxiety, substance addiction, obsessive-compulsive disorder, etc.

[0004] Psilocybe L-tryptophan-specific decarboxylase PsiD belongs to the phosphatidylserine decarboxylase family of proteins, and its structure can generate two chains after self-cleavage between the "GGSS" (G401-G402-S403-S404) sequences to play a decarboxylation role. In the Psilocybin biosynthesis pathway, the psiD protein catalyzes the generation of tryptamine using L-tryptophan as a substrate, or decarboxylates 4-hydroxy-L-tryptophan as a substrate to generate 4-hydroxy-tryptamine. The present invention helps to clarify its decarboxylation mechanism in the Psilocybin biosynthesis process and the self-inhibition mechanism at the N-terminal W27 position by analyzing the three-dimensional crystal structure of the Psilocybin PsiD (50-439) truncated protein complex, thereby better providing biotechnological guidance for Psilocybin biosynthesis and providing more possibilities for increasing the yield of Psilocybin. Summary of the invention

[0005] The purpose of the present invention is to provide a three-dimensional crystal structure of a truncated Psilocybin PsiD protein and a tryptamine complex, which helps to clarify the decarboxylation mechanism of the Psilocybin PsiD protein in the Psilocybin biosynthesis process and the autoinhibition mechanism at the N-terminal W27 position, thereby better providing biotechnological guidance for Psilocybin biosynthesis and providing more possibilities for increasing the yield of Psilocybin.

[0006] The present invention also provides a crystallization method and application of a complex of a truncated Psilocybin PsiD protein and tryptamine.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a three-dimensional crystal structure of a complex of a Psilocybin PsiD protein truncate and tryptamine, wherein the complex comprises a Psilocybin PsiD protein truncate and tryptamine, wherein the Psilocybin PsiD protein truncate has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity thereto;

[0009] 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

[0010] Furthermore, the resolution of the three-dimensional crystal structure of the complex reaches

[0011] The space group of the three-dimensional crystal structure of the complex is P2 1 ;

[0012] The unit cell parameters of the three-dimensional crystal structure of the complex are: α=90°, β=93.46°, γ=90°.

[0013] Furthermore, the three-dimensional crystal structure of the complex contains two truncated Psilocybin PsiD proteins and two tryptamines in one crystallographic asymmetric unit, and the secondary structure of the truncated Psilocybin PsiD protein in the three-dimensional crystal structure of the complex includes 16 α-helices and 13 β-folds;

[0014] Among them, α helix 1, i.e., the amino acid segment containing Glu59-Asp70; α helix 2, i.e., the amino acid segment containing Pro72-Phe82; α helix 3, i.e., the amino acid segment containing Try92-Arg104; α helix 4, i.e., the amino acid segment containing Pro115-Ans125; α helix 5, i.e., the amino acid segment containing Arg127-Thr134; α helix 6, i.e., the amino acid segment containing Gln136-Ser154; α helix 7, i.e., the amino acid segment containing Lys156-Val 161; α helix 8, i.e., the amino acid segment containing Glu177-Val 184; α helix 9, i.e., the amino acid segment containing Phe192-Val α helix 10, i.e., the amino acid segment containing Tyr210-Ans215; α helix 11, i.e., the amino acid segment containing Arg221-Asp224; β fold 1, i.e., the amino acid segment containing Leu236-Ala239; β fold 2, i.e., the amino acid segment containing Ser243-Try250; α helix 12, i.e., the amino acid segment containing Leu267-Leu271; α helix 13, i.e., the amino acid segment containing Phe276-Phe280; β fold 3, i.e., the amino acid segment containing Ser284-Phe289; β fold 4, i.e., the amino acid segment containing Arg297-His299; β fold 5, i.e., the amino acid segment containing Gly304-Val312; α helix 14, i.e., the amino acid segment containing Phe321-Thr323; β-fold 11, i.e., the amino acid segment containing Gly404-Leu409; α-helix 16, i.e., the amino acid segment containing Glu419-Thr422; β-fold 12, i.e., the amino acid segment containing Gly425-Ile428; β-fold 13, i.e., the amino acid segment containing Val434-Ala436.

[0015] Furthermore, in the three-dimensional crystal structure of the complex, due to the self-cleavage mechanism of "GGSS", two chains can be generated after self-cleavage between the G401-G402-S403-S404 sequences to exert a decarboxylation effect. The pyruvyl group is formed by the splitting of G402 and S403 at the N-terminus of the α chain. The methyl group of the pyruvyl group interacts with Phe398, His296 and Leu290; the covalent amide forms a hydrogen bond with the main chain of Leu290; the hydrogen on the indole ring of the tryptamine is bound to the side chain of Thr374 and the main chain of Met373; the indole ring is surrounded by Phe289, Val292, Leu339, Leu112, Tyr338, Tyr117, Pro114, Met373, Thr374 and Ile376, thereby stabilizing the intermediate product.

[0016] Based on the same inventive concept, the present invention provides a method for crystallizing a complex of a truncated Psilocybin Ps iD protein and tryptamine, the crystallization method comprising:

[0017] The truncated Psilocybin Ps iD protein solution was concentrated to 10 mg / ml, then L-tryptophan was added to dissolve it, and incubated on ice for 60 min to obtain a mixed solution;

[0018] Using crystallization reagents as the initial screening conditions for crystal growth, crystallization was performed by sitting drop or hanging drop vapor diffusion method at a crystallization temperature of 18°C ​​to obtain crystals of the complex of Psilocybin Ps iD protein truncations and tryptamines;

[0019] Wherein, the truncated form of the Psilocybin Ps iD 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] In the mixed solution, the molar ratio of the Psilocybin Ps iD protein truncate to the L-tryptophan is 1:10.

[0021] Furthermore, the volume ratio of the mixed solution to the crystallization reagent is 3:1 to 1:3;

[0022] The crystallization is carried out by sitting drop and hanging drop vapor diffusion method, and the crystallization conditions are 0.1M magnesium formate dihydrate and 15% w / v polyethylene glycol 3350.

[0023] Based on the same inventive concept, the present invention provides a method for expressing and purifying a truncated form of the Psilocybin mushroom Ps iD protein, the expression and purification method comprising:

[0024] Constructing a fusion plasmid of the coding gene of the Psilocybin mushroom Ps iD protein truncation and pET28a-Sumo, transforming the fusion plasmid into Escherichia coli, and expressing the Psilocybin mushroom Ps iD protein truncation containing a specific tag;

[0025] The Psilocybe occidentalis Ps iD protein truncated body containing the specific tag is separated by a method that specifically recognizes the specific tag, the Psilocybe occidentalis Ps iD protein truncated body is separated by Ni affinity chromatography after the specific tag is cleaved, and then a high-purity Psilocybe occidentalis Ps iD protein truncated body is obtained by gel filtration chromatography;

[0026] Wherein, the Escherichia coli includes Escherichia coli BL21 (DE3);

[0027] The truncated version of the Psilocybin Ps iD protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity therewith.

[0028] Furthermore, the construction of a fusion plasmid of the coding gene of the Psilocybin mushroom Ps iD protein truncation and pET28a-Sumo, transforming the fusion plasmid into Escherichia coli, and expressing the Psilocybin mushroom Ps iD protein truncation containing a specific tag specifically includes:

[0029] The coding gene of the Psilocybin mushroom PsiD protein truncation was cloned into the pET28a-Sumo plasmid, and the N-terminus of the coding gene of the Psilocybin mushroom PsiD protein truncation was tagged with a His6-SUMO tag to obtain a fusion plasmid;

[0030] The fusion plasmid was transformed into Escherichia coli to express the Psilocybin Ps iD protein truncated form containing the His6-SUMO tag.

[0031] Based on the same inventive concept, the present invention provides the application of the three-dimensional crystal structure of the complex of Psilocybin PsiD protein truncations and tryptamine in guiding and regulating the decarboxylation step of the Psilocybin biosynthesis process.

[0032] Based on the same inventive concept, the present invention provides the application of the three-dimensional crystal structure of the complex of Psilocybin PsiD protein truncations and tryptamine in guiding the improvement of Psilocybin biosynthesis yield or synthesis efficiency.

[0033] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0034] The three-dimensional crystal structure of the complex of the Psilocybe occidentalis PsiD protein truncated body and tryptamine of the present invention, wherein the complex comprises the Psilocybe occidentalis PsiD protein truncated body and tryptamine, wherein the Psilocybe occidentalis PsiD protein truncated body contains 390 amino acids and has a molecular weight of 44.33 kDa, and the three-dimensional structure of the complex of the Psilocybe occidentalis PsiD protein truncated body and tryptamine is obtained by X-ray diffraction with a resolution of Its space group is P2 1In the crystal structure of the complex, the secondary structure of the Psilocybe PsiD protein is composed of 16 α helices and 13 β folds. Its structure undergoes self-cleavage between the "GGSS" (G401-G402-S403-S404) motifs to produce α and β chains, and shows an autoinhibitory mechanism at the N-terminal W27 position. In the process of psilocybin biosynthesis, the PsiD protein catalyzes the generation of tryptamine using L-tryptophan as a substrate, or decarboxylates 4-hydroxy-L-tryptophan as a substrate to generate 4-hydroxy-tryptamine. Therefore, the analysis of the three-dimensional crystal structure of the PsiD (50-439) truncation protein complex helps to clarify its decarboxylation mechanism in the process of psilocybin biosynthesis, as well as the autoinhibitory mechanism at the N-terminal W27 position, thereby providing better structural basis guidance for psilocybin biosynthesis and providing more possibilities for increasing the yield of psilocybin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 The results of gel filtration chromatography and SDS-PAGE detection of PsiD (50-439) truncated protein.

[0037] Figure 2 This is a crystal picture of the complex between PsiD (50-439) truncated protein and tryptamine.

[0038] Figure 3 Schematic diagram of the three-dimensional structure of the complex between PsiD (50-439) truncated protein and tryptamine: (A) Schematic diagram of the three-dimensional structure of the complex between PsiD (50-439) truncated protein and tryptamine; (B) Surface charge distribution of the complex between PsiD (50-439) truncated protein and tryptamine.

[0039] Figure 4 A, B, and C are comparisons of the self-cleavage efficiency and product amount of PsiD (50-439) truncated protein with the full-length, PsiD (50-439) W27A mutant, and PsiD (30-439) truncate protein.

[0040] Figure 5 This is the plasmid map of the recombinant vector. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The three-dimensional crystal structure, crystallization method and application of the complex of the truncated Psilocybin PsiD protein and tryptamine of the present application will be described in detail below in combination with the examples and experimental data.

[0045] Example 1

[0046] The expression and purification of Psilocybin PsiD (Asn50-Ala439) truncated protein are as follows:

[0047] 1) Construct the plasmid of Psilocybe PsiD gene, use wild-type PsiD as template, clone Asn50-Ala439 fragment by PCR, clone Psilocybe PsiD (Asn50-Ala439) gene into pET28a-Sumo vector by molecular cloning technology, and use it to express the fusion protein with N-terminal fusion His6-SUMO tag to obtain the recombinant vector (see vector plasmid map for details). Figure 5 ).

[0048] The nucleotide sequence of the gene encoding PsiD (Asn50-Ala439) of Psilocybin mushrooms is shown in SEQ ID NO. 2. The upstream primer used for PCR amplification of the Psilocybin mushroom PsiD (Asn50-Ala439) gene is: 5'-CACAGGATCCAATTACTCCAACGAGTTCGGCCTC-3', and the downstream primer is: 5'-GACACTCGAGCTAAGCCTTTAGAGCAGCGACGA-3'.

[0049] 2) Transform the recombinant vector into the E. coli BL21 (DE3) strain (the truncated protein is highly soluble in the E. coli BL21 (DE3) strain), add kanamycin sulfate to 1L of LB liquid medium to make the final concentration of 50ug / ml, and continue to shake culture at 37°C and 220rpm until the OD reaches 0.6-0.8 (about 4h), then add 200ul1M IPTG to each liter of culture to make the final concentration of IPTG 0.2mM, and finally induce overnight at 18°C ​​and 220rpm for 16-20h. Next, collect the induced expression of the bacterial solution in a bacterial collection bucket, centrifuge at 4000rpm and 4°C for 10min, discard the supernatant, precipitate the bacteria, collect in a 50ml centrifuge tube, resuspend and wash the bacteria with 20mL 1x PBS. After the bacteria are added to the resuspension solution and resuspended, centrifuge at 4000rpm and 4°C for 15min, discard the supernatant, and harvest the bacterial precipitate for protein purification.

[0050] 3) Add 5 times the volume of the balance buffer (containing 20mM Tris8.0, 500mM NaCl, 25mM imidazole; the same below) of the bacteria (the bacteria concentration is easiest to break between 10% and 20%), and blow thoroughly to resuspend the bacteria. Precool, clean, and rinse with buffer in the high-pressure crusher, and repeatedly crush the bacteria at 1200-1500bar for 3-4 times (after the crushing, it should flow out like water and not be viscous). Transfer the lysate to a high-speed centrifuge tube and centrifuge at 17000rpm and 4℃ for 1 hour. After centrifugation, discard the precipitate and transfer the supernatant to a 50ml centrifuge tube.

[0051] 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:

[0052] The supernatant of high-speed centrifugation was loaded onto a cleaned and equilibrated nickel column (5mL HisTrap pre-packed column) at a 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 Tris8.0, 500mM NaCl, 500mM imidazole; the same below)". When the A280 value began to increase, protein collection began 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, 500mM NaCl,) and dialyzed at 4℃ for 3 hours. The collected protein solution was loaded onto two series-connected cleaned and equilibrated nickel columns at a rate of 1ml / min. The flow-through liquid was detected by Bradford. When the Bradford color began to change, the flow-through liquid was collected immediately. After the protein sample was loaded, the buffer solution continued to flow through the column until Bradford detected very little protein in the flow-through liquid.

[0053] 5) Next, the collected protein was concentrated using a 10 kDa cutoff concentrator, and the concentrated protein was further purified by gel filtration chromatography (chromatographic column using Superdex 200 16 / 600. Stovan, 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.

[0054] In this embodiment, the prokaryotic cell expression system of Escherichia coli is preferably used (but other expression systems, such as expression in other prokaryotic cells or eukaryotic cells are not excluded), and the above-mentioned protein is expressed in the form of a fusion protein, and the His tag is preferably selected to obtain soluble expression (but other tags, such as MBP or GST tags, are also excluded, which can also achieve soluble expression).

[0055] Example 2

[0056] The method for crystallizing the complex of Psilocybin PsiD (50-439) truncated protein and L-tryptophan comprises:

[0057] 1) A PsiD (50-439) protein concentration of 10 mg / ml was preferentially selected for crystal growth, and the protein of this concentration was mixed with L-tryptophan at a molar ratio of 1:10, and incubated on ice for 60 minutes to obtain a mixed solution.

[0058] 2) Using the crystallization reagent in the crystallization kit (Index, Crystal Screen Ⅰ / Ⅱ, JCSG Core Ⅰ-Ⅳ Suite, etc. from Hampton Research and other companies) as the initial screening conditions for crystal growth, and optimizing the initial screening conditions by hanging drop, crystallization is carried out by sitting drop vapor diffusion method, and 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.

[0059] The present invention obtains initial crystals under multiple different crystallization reagent conditions. Through subsequent optimization and adjustment, 0.1M magnesium formate dihydrate and 15% w / v polyethylene glycol 3350 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.

[0060] Specifically, the three-dimensional crystal structure of the complex of the LL-tryptophan-specific decarboxylase PsiD (50-439) truncated protein and tryptamine of Psilocybin mushroom has two PsiD (50-439) protein molecules in one crystallographic asymmetric unit, one of which is composed of 16 α-helices and 13 β-folds. Figure 3 As shown in A.

[0061] Among them, α helix 1, i.e., the amino acid segment containing Glu59-Asp70; α helix 2, i.e., the amino acid segment containing Pro72-Phe82; α helix 3, i.e., the amino acid segment containing Try92-Arg104; α helix 4, i.e., the amino acid segment containing Pro115-Ans125; α helix 5, i.e., the amino acid segment containing Arg127-Thr134; α helix 6, i.e., the amino acid segment containing Gln136-Ser154; α helix 7, i.e., the amino acid segment containing Lys156-Val161; α helix 8, i.e., the amino acid segment containing Glu1 77-Val184; α helix 9, i.e., the amino acid segment containing Phe192-Val195; α helix 10, i.e., the amino acid segment containing Tyr210-Ans215; α helix 11, i.e., the amino acid segment containing Arg221-Asp224; β fold 1, i.e., the amino acid segment containing Leu236-Ala239; β fold 2, i.e., the amino acid segment containing Ser243-Try250; α helix 12, i.e., the amino acid segment containing Leu267-Leu271; α helix 13, i.e., the amino acid segment containing Phe276-Ph β-fold 3, i.e., the amino acid segment containing Ser284-Phe289; β-fold 4, i.e., the amino acid segment containing Arg297-His299; β-fold 5, i.e., the amino acid segment containing Gly304-Val312; α-helix 14, i.e., the amino acid segment containing Phe321-Thr323; α-helix 15, i.e., the amino acid segment containing Phe337-Ile348; β-fold 6, i.e., the amino acid segment containing Arg351-Ala358; β-fold 7, i.e., the amino acid segment containing Leu365-Ile371; The β-fold 8 is an amino acid segment containing Thr378-Ala381; β-fold 9 is an amino acid segment containing Gly386-Val389; β-fold 10 is an amino acid segment containing Leu395-His399; β-fold 11 is an amino acid segment containing Gly404-Leu409; α-helix 16 is an amino acid segment containing Glu419-Thr422; β-fold 12 is an amino acid segment containing Gly425-Ile428; β-fold 13 is an amino acid segment containing Val434-Ala436. The crystal structure of the entire complex contains the mechanism of PsiD-catalyzed decarboxylation (including the self-cleavage mechanism) and the self-inhibition mechanism that occurs when Trp27 on the N-terminal chain competes with the substrate for the binding pocket.

[0062] Its structural feature is the "GGSS" self-cleavage mechanism. After self-cleavage between the G401-G402-S403-S404 sequence, two chains can be generated to play a decarboxylation role. The pyruvyl group is formed by the cleavage of G402 and S403 at the N-terminus of the α chain. The methyl group of pyruvyl interacts with Phe398, His296 and Leu290; the covalent amide forms a hydrogen bond with the main chain of Leu290; the hydrogen on the indole ring of tryptamine is combined with the side chain of Thr374 and the main chain of Met373; the indole ring is surrounded by Phe289, Val292, Leu339, Leu112, Tyr338, Tyr117, Pro114, Met373, Thr374 and Ile376, thereby stabilizing the intermediate product.

[0063] The surface charge distribution of the complex between PsiD(50-439) truncated protein and tryptamine is as follows Figure 3 As shown in B, the substrate binding pocket is located in the deep cleft region and is surrounded by negative charges.

[0064] This three-dimensional crystal structure describes the secondary structure composition of the Ps iD (50-349) protein, the direction of the peptide chain, the organizational pattern, the three-dimensional molecular structure, its binding mode with tryptamine, the self-cleavage mechanism of the GGSS sequence, the decarboxylation mechanism, and the self-inhibition mechanism of the Trp27 position.

[0065] The truncated Ps iD protein of Psilocybe occidentalis undergoes self-cleavage between the "GGSS" (G401-G402-S403-S404) motif to produce α chain and β chain, and Trp27 at the N-terminus competes with the substrate for the binding pocket, resulting in autoinhibition.

[0066] The atomic coordinate groups of the three-dimensional crystal structure of the complex of Psilocybin Ps iD (50-439) truncated protein and tryptamine are as follows:

[0067] Crystal space group: P2 1

[0068] The unit cell parameters are: α=90°, β=93.46°, γ=90°.

[0069] I / σI: 20.4(3.5)

[0070] Rwork and Rfree are: 0.1649 / 0.1810

[0071] Resolution range 50.00-1.60

[0072] Data completeness (%): 99.9 (100%)

[0073] Table 1

[0074]

[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]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Data collection and correction statistics

[0213]

[0214]

[0215] *Highest resolution included in brackets.

[0216] Example 3

[0217] (1) To verify the autoinhibitory effect of the Trp27 position of the truncated protein of Psilocybin Ps iD (50-439), Trp27 was mutated to alanine. At the same time, two truncated proteins, Ps iD (30-439) and Ps iD (50-439), were made to prove that the N-terminal 50 amino acids have no effect on the auto-cleavage reaction. Removing the above autoinhibitory effect can enhance the enzyme activity.

[0218] Ps iD (1-439), Ps iD (30-439), Ps iD (50-439) and mutant Ps iD (1-439) W27A were purified and run on SDS-PAGE gel to prove that the N-terminal 50 amino acids do not affect self-cleavage. Figure 4 As shown in B, there is no significant difference in the self-cleavage activity (producing α chain) between the truncated forms and mutants and WT.

[0219] (2) Ps iD (1-439), Ps iD (30-439), Ps iD (50-439) and mutant Ps iD (1-439) W27A were mixed with substrate L-tryptophan at a molar ratio of 1:400, incubated at 37°C for 14 minutes, and then developed by thin layer chromatography (20 ml of developing agent was poured into one side of a glass cylinder, and the chromatographic silica gel plate was pre-saturated in the cylinder for 1 hour after being spotted with a capillary tube, and developed for 3 hours) to detect the yield of the reaction product tryptamine. Figure 4 As shown in A, the amount of product generated by the mutant and truncation was significantly increased compared with the WT. The results of the truncation indicated that there was autoinhibition at the N-terminus, and the results of the mutant proved that the Trp27 position would produce autoinhibition on the Ps iD active site.

[0220] (3) The amount of tryptamine product generated was calculated by taking the gray value using Image J after three repeated experiments: Ps iD (50-439) > Ps iD (30-439) > Ps iD (1-439) W27A > Ps iD (1-439) WT. Figure 4 As shown in C.

[0221] 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.

[0222] 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.

[0223] 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 between the truncated PsiD protein of Psilocybin and tryptamine, characterized in that: The complex comprises a truncated PsiD protein of Psilocybin and tryptamine, wherein the truncated PsiD protein of Psilocybin 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 PsiD protein truncations and tryptamine 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 P21; The unit cell parameters of the three-dimensional crystal structure of the complex are: α=90°, β=93.46°, γ=90°.

3. The three-dimensional crystal structure of the complex of Psilocybin PsiD protein truncations and tryptamine according to claim 1, characterized in that: The three-dimensional crystal structure of the complex contains two Psilocybin PsiD protein truncations and two tryptamines in one crystallographic asymmetric unit, and the secondary structure of the Psilocybin PsiD protein truncations in the three-dimensional crystal structure of the complex includes 16 α-helices and 13 β-folds; Among them, α helix 1, i.e., the amino acid segment containing Glu59-Asp70; α helix 2, i.e., the amino acid segment containing Pro72-Phe82; α helix 3, i.e., the amino acid segment containing Try92-Arg104; α helix 4, i.e., the amino acid segment containing Pro115-Ans125; α helix 5, i.e., the amino acid segment containing Arg127-Thr134; α helix 6, i.e., the amino acid segment containing Gln136-Ser154; α helix 7, i.e., the amino acid segment containing Lys156-Val 161; α helix 8, i.e., the amino acid segment containing Glu177-Val 184; α helix 9, i.e., the amino acid segment containing Phe192-Val α helix 10, i.e., the amino acid segment containing Tyr210-Ans215; α helix 11, i.e., the amino acid segment containing Arg221-Asp224; β fold 1, i.e., the amino acid segment containing Leu236-Ala239; β fold 2, i.e., the amino acid segment containing Ser243-Try250; α helix 12, i.e., the amino acid segment containing Leu267-Leu271; α helix 13, i.e., the amino acid segment containing Phe276-Phe280; β fold 3, i.e., the amino acid segment containing Ser284-Phe289; β fold 4, i.e., the amino acid segment containing Arg297-His299; β fold 5, i.e., the amino acid segment containing Gly304-Val312; α helix 14, i.e., the amino acid segment containing Phe321-Thr323; β-fold 11, i.e., the amino acid segment containing Gly404-Leu409; α-helix 16, i.e., the amino acid segment containing Glu419-Thr422; β-fold 12, i.e., the amino acid segment containing Gly425-Ile428; β-fold 13, i.e., the amino acid segment containing Val434-Ala436.

4. A method for crystallizing a complex of a truncated PsiD protein of Psilocybin and tryptamine, characterized in that: The crystallization method comprises: The truncated PsiD protein solution of Psilocybin mushroom was concentrated to 10 mg / ml, then L-tryptophan was added to dissolve it, and incubated on ice for 60 min to obtain a mixed solution; Using crystallization reagents as the initial screening conditions for crystal growth, crystallization was performed by sitting drop or hanging drop vapor diffusion method at a crystallization temperature of 18°C ​​to obtain crystals of the complex of Psilocybin PsiD protein truncations and tryptamines; Wherein, the truncated form of the Psilocybin mushroom PsiD 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 PsiD protein truncate to the L-tryptophan is 1:

10.

5. The method for crystallizing the complex of Psilocybin PsiD protein truncations and tryptamine according to claim 4, 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 and hanging drop vapor diffusion method, and the crystallization conditions are 0.1M magnesium formate dihydrate and 15% w / v polyethylene glycol 3350.

6. A method for expressing and purifying a truncated Psilocybin protein PsiD, characterized in that: The expression and purification method comprises: Constructing a fusion plasmid of the coding gene of the Psilocybin mushroom PsiD protein truncation and pET28a-Sumo, transforming the fusion plasmid into Escherichia coli, and expressing the Psilocybin mushroom PsiD protein truncation containing a specific tag; The Psilocybe occidentalis PsiD protein truncations containing the specific tag are separated by a method that specifically recognizes the specific tag, the specific tag is cleaved by enzyme, the Psilocybe occidentalis PsiD protein truncations are separated by Ni affinity chromatography, and then a high-purity Psilocybe occidentalis PsiD protein truncations are obtained by gel filtration chromatography; Wherein, the Escherichia coli includes Escherichia coli BL21 (DE3); The truncated Psilocybin PsiD protein has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80% sequence identity therewith.

7. The method for expressing and purifying a truncated Psilocybin protein of claim 6, characterized in that: The construction of a fusion plasmid of a gene encoding a truncated Psilocybin mushroom PsiD protein and pET28a-Sumo, transforming the fusion plasmid into Escherichia coli, and expressing a truncated Psilocybin mushroom PsiD protein containing a specific tag specifically comprises: The coding gene of the Psilocybin mushroom PsiD protein truncation was cloned into the pET28a-Sumo plasmid, and the N-terminus of the coding gene of the Psilocybin mushroom PsiD protein truncation was tagged with a His6-SUMO tag to obtain a fusion plasmid; The fusion plasmid was transformed into Escherichia coli to express the Psilocybin PsiD protein truncated form containing the His6-SUMO tag.

8. Use of the three-dimensional crystal structure of the complex of the Psilocybin PsiD protein truncated form and tryptamine according to any one of claims 1 to 3 in guiding and regulating the decarboxylation step of the Psilocybin biosynthesis process.

9. Use of the three-dimensional crystal structure of the complex of Psilocybin PsiD protein truncations and tryptamine according to any one of claims 1 to 3 in guiding the improvement of Psilocybin biosynthesis yield or synthesis efficiency.

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