HsSAHH enzyme variants, nucleic acids, expression vectors, their applications, and kits

Abstract

This invention provides an HsSAHH enzyme variant, a kit, and its applications. Compared to the wild-type HsSAHH shown in SEQ ID NO: 1, this variant contains one or more amino acid modifications in the metal coordination motif of wild-type HsSAHH. Compared to wild-type HsSAHH, this variant exhibits significantly increased soluble expression yield and significantly higher specific enzyme activity, thus making it more suitable for the quantitative detection of homocysteine.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, specifically to the protein engineering modification of human S-adenosyl homocysteine ​​hydrolase (HsSAHH), and particularly to an HsSAHH enzyme variant and its application in homocysteine ​​detection. Background Technology

[0002] Homocysteine ​​(Hcy) is a sulfur-containing amino acid produced by the metabolism of methionine and is widely recognized as an important clinical biomarker. Quantitative analysis of Hcy in clinical diagnosis typically involves the use of S-adenosylhomocysteine ​​hydrolase (SAHH).

[0003] To address the insufficient activity and expression levels of SAHH enzymes in existing technologies, various improvement strategies have been developed. For example, some studies have successfully increased the recombinant expression yield of SAHH derived from *Sulphurobacterium* by truncating its N-terminus; other methods enhance SAHH activity through exogenous supplementation of the coenzyme NAD⁺; and cation-mediated regulation has also been used to optimize the catalytic performance of SAHH. However, these improvement strategies either fail to solve the fundamental problem of low catalytic turnover, are not economically feasible for large-scale diagnostics due to the high cost of NAD⁺, or their overall performance still cannot meet the stringent requirements of industrial-scale processes.

[0004] Human S-adenosylhomocysteine ​​hydrolase (HsSAHH, UniProtKB P23526) is a key enzyme widely used in homocysteine ​​detection kits based on enzyme cycling assays. However, wild-type HsSAHH suffers from low soluble expression yield and insufficient catalytic activity, limiting its performance in diagnostic applications. Therefore, there is an urgent need in the field to provide a variant of human HsSAHH that can simultaneously improve its soluble expression level and catalytic activity, in order to meet the comprehensive requirements of homocysteine ​​detection kits for diagnostic accuracy, specificity, and cost-effectiveness. Summary of the Invention

[0005] The main objective of this invention is to provide an HsSAHH enzyme variant, a kit, and its applications, thereby solving the problems of low soluble expression yield and insufficient specific enzyme activity of wild-type HsSAHH in the prior art.

[0006] The first aspect of the present invention provides an HsSAHH enzyme variant that, compared with the wild-type HsSAHH shown in SEQ ID NO: 1, contains one or more amino acid modifications in the metal coordination motif of the wild-type HsSAHH.

[0007] Furthermore, the metal coordination motif includes a first metal coordination motif and a second metal coordination motif; the first metal coordination motif is amino acids at positions 55-59; and the second metal coordination motif is amino acids at positions 351-355.

[0008] Furthermore, the amino acid modification includes a first amino acid modification and / or a second amino acid modification; the first amino acid modification is to mutate a specific amino acid in the first metal ligand motif, and the second amino acid modification is to mutate a specific amino acid in the second metal ligand motif.

[0009] Furthermore, the first amino acid modification involves mutating amino acid position 55 to glutamine; the second amino acid modification involves mutating amino acid position 351 to threonine.

[0010] Furthermore, the variant is selected from one of the following: 1) E59Q variant; 2) M351T variant; 3) E59Q / M351T variant.

[0011] A second aspect of the present invention provides a nucleic acid that encodes any of the above-mentioned HsSAHH enzyme variants.

[0012] A third aspect of the present invention provides an expression vector comprising the above-described nucleic acid.

[0013] The fourth aspect of this invention provides the application of the above-mentioned HsSAHH enzyme variant in homocysteine ​​detection reagents.

[0014] The fifth aspect of the present invention provides a homocysteine ​​detection kit comprising any of the above-mentioned SAHH enzyme variants.

[0015] Furthermore, the kit also includes S-adenosylmethionine.

[0016] By applying the technical solution of this invention, site-specific amino acid modification of the metal coordination motif of the wild-type HsSAHH enzyme significantly improves the soluble expression yield of the HsSAHH enzyme variant in the recombinant expression system. Simultaneously, the amino acid modification significantly enhances the specific activity of the variant compared to the wild-type SAHH, further improving the enzyme's catalytic efficiency. Attached Figure Description

[0017] Figure 1 During a 300 ns molecular dynamics simulation at 310 K, the RMSD of (a) wild-type HsSAHH and (b) E59Q / M351T mutants was measured.

[0018] Figure 2 (a) Nucleophilic attack distance between K186-Nζ and SAH-3'-OH in wild-type HsSAHH and (b) E59Q / M351T mutants. The magenta dashed line indicates the nucleophilic attack distance. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0020] As mentioned in the background section, existing technologies exhibit low expression yield and insufficient catalytic activity of wild-type HsSAHH enzymes. Therefore, in this application, the inventors have attempted to develop an HsSAHH enzyme variant, a kit, and its applications to improve the expression yield and specific activity of the HsSAHH enzyme, thereby enhancing its catalytic efficiency. Based on this, the applicant has proposed a series of protection schemes for this application.

[0021] In a typical embodiment, an HsSAHH enzyme variant is provided, which, compared to the wild-type HsSAHH shown in SEQ ID NO: 1, includes one or more amino acid modifications in the metal coordination motif of the wild-type HsSAHH. By site-specifically modifying the metal coordination motif with amino acids, the enzyme expression yield and catalytic activity are improved, thereby enhancing the catalytic efficiency of the variant.

[0022] SEQ ID NO:1: MSDKLPYKVADIGLAAWGRKALDIAENEMPGLMRMRERYSASKPLKGARIAGCLHMTVETAVLIETLVTLGAEVQWSSCNIFSTQDHAAAAIAKAGIPVYAWKGETDE EYLWCIEQTLYFKDGPLNMILDDGGDLTNLIHTKYPQLLPGIRGISEETTTGVHNLYKMMANGILKVPAINVNDSVTKSKFDNLYGCRESLIDGIKRATDVMIAGKVA VVAGYGDVGKGCAQALRGFGARVIITEIDPINALQAAMEGYEVTTMDEACQEGNIFVTTTGCIDIILGRHFEQMKDDAIVCNIGHFDVEIDVKWLNENAVEKVNIKPQ VDRYRLKNGRRIILLAEGRLVNLGCAMGHPSFVMSNSFTNQVMAQIELWTHPDKYPVGVHFLPKKLDEAVAEAHLGKLNVKLTKLTEKQAQYLGMSCDGPFKPDHYRY

[0023] The wild-type HsSAHH described in this article is a human S-adenosylhomocysteine ​​hydrolase, a key metabolic enzyme widely distributed in organisms that catalyzes the hydrolysis of S-adenosylhomocysteine ​​to adenosine and homocysteine. This enzyme plays a central regulatory role in the methylation metabolic pathway, maintaining the dynamic balance of intracellular S-adenosylmethionine and homocysteine. HsSAHH is a homotetrameric protein, with each subunit having a molecular weight of approximately 48 kDa. Its amino acid sequence is shown in SEQ ID NO: 1, which is a well-known wild-type HsSAHH sequence obtained from UniProtKB, P23526.

[0024] The "amino acid modification" mentioned in this article refers to the replacement of amino acid residues at specific positions in wild-type SAHH enzymes with other amino acid residues using site-directed mutagenesis, including "amino acid mutation" or "amino acid substitution". It is usually represented using the format "X position Y", where X is a single-letter or three-letter abbreviation of the original amino acid, the position is the amino acid's position number in the sequence, and Y is a single-letter or three-letter abbreviation of the substituted amino acid. For example, "E59Q" indicates that glutamic acid (Glu, E) at position 59 is replaced by glutamine (Gln, Q).

[0025] The “metal coordination motif” mentioned in this article refers to a conserved amino acid sequence in the SAHH protein that participates in the binding of metal ions. This motif plays an important role in maintaining the enzyme’s spatial conformation, catalytic activity, and substrate binding ability.

[0026] The term "soluble expression yield" as used in this article refers to the amount of recombinant protein expressed in a soluble form in host cells and recoverable from cell lysate supernatant, expressed as the amount of protein per unit volume of culture medium (mg / L). Increased soluble expression yield means that more proteins with native conformation and activity are available, which helps reduce production costs.

[0027] The term "specific activity" as used in this article refers to the enzyme catalytic activity per unit mass of protein, usually expressed in U / mg. Higher specific activity indicates higher catalytic efficiency per unit mass of protein, requiring less enzyme to be added to the detection system, which helps reduce reagent costs and minimizes non-specific interference.

[0028] The "catalytic efficiency" mentioned in this article refers to the ability of an enzyme to catalyze the conversion of a substrate into a product, usually expressed as k_cat / K_m. Higher catalytic efficiency indicates a stronger affinity of the enzyme for the substrate, a faster conversion rate, and consequently, a faster signal response and higher detection sensitivity in the detection system.

[0029] In one specific embodiment, the metal coordination motif includes a first metal coordination motif and a second metal coordination motif; the first metal coordination motif is an amino acid at positions 55-59; and the second metal coordination motif is an amino acid at positions 351-355.

[0030] HsSAHH comprises a substrate-binding domain, a cofactor-binding domain, and a C-terminal dimerization domain. In the substrate-binding domain, the first metal coordination motif, residues 55–59 of HsSAHH, interacts with the purine ring of the substrate; this contact directly participates in the catalytic process. The second metal coordination motif, residues 351–355 of HsSAHH, is located in the hinge region connecting the substrate-binding domain and the cofactor-binding domain. Studies have shown that metal ions mediate substrate-induced conformational changes by regulating the orientation of amino acid side chains, thereby optimizing ligand interactions and ultimately regulating catalytic efficiency and substrate affinity. Therefore, the first and second metal coordination motifs are correlated with the catalytic performance of HsSAHH.

[0031] In one specific embodiment, the amino acid modification includes a first amino acid modification and / or a second amino acid modification; the first amino acid modification involves mutating a specific amino acid in a first metal coordination motif, and the second amino acid modification involves mutating a specific amino acid in a second metal coordination motif. Preferably, the first amino acid modification involves mutating amino acid position 55 to glutamine; and the second amino acid modification involves mutating amino acid position 351 to threonine.

[0032] In one specific implementation, the variant is selected from one of the following: 1) E59Q variant; 2) M351T variant; 3) E59Q / M351T variant; The E59Q variant is the protein shown in SEQ ID NO: 2; the M351T variant is the protein shown in SEQ ID NO: 3; and the E59Q / M351T variant is the protein shown in SEQ ID NO: 4.

[0033] SEQ ID NO:2: MSDKLPYKVADIGLAAWGRKALDIAENEMPGLMRMRERYSASKPLKGARIAGCLHMTVQTAVLIETLVTLGAEVQWSSCNIFSTQDHAAAAIAKAGIPVYAWKGETDEEYLWCIEQTLYFKDGPLNMILDDGGDLTNLIHTKYPQLLPGIRGISEETTTGVHNLYKMMANGILKVPAINVNDSVTKSKFDNLYGCRESLIDGIKRATDVMIAGKVAVVAGYGDVGKGCAQALRGFGARVIITEIDPINALQAAMEGYEVTTMDEACQEGNIFVTTTGCIDIILGRHFEQMKDDAIVCNIGHFDVEIDVKWLNENAVEKVNIKPQVDRYRLKNGRRIILLAEGRLVNLGCAMGHPSFVMSNSFTNQVMAQIELWTHPDKYPVGVHFLPKKLDEAVAEAHLGKLNVKLTKLTEKQAQYLGMSCDGPFKPDHYRY SEQ ID NO:3: MSDKLPYKVADIGLAAWGRKALDIAENEMPGLMRMRERYSASKPLKGARIAGCLHMTVETAVLIETLVTLGAEVQWSSCNIFSTQDHAAAAIAKAGIPVYAWKGETDEEYLWCIEQTLYFKDGPLNMILDDGGDLTNLIHTKYPQLLPGIRGISEETTTGVHNLYKMMANGILKVPAINVNDSVTKSKFDNLYGCRESLIDGIKRATDVMIAGKVAVVAGYGDVGKGCAQALRGFGARVIITEIDPINALQAAMEGYEVTTMDEACQEGNIFVTTTGCIDIILGRHFEQMKDDAIVCNIGHFDVEIDVKWLNENAVEKVNIKPQVDRYRLKNGRRIILLAEGRLVNLGCATGHPSFVMSNSFTNQVMAQIELWTHPDKYPVGVHFLPKKLDEAVAEAHLGKLNVKLTKLTEKQAQYLGMSCDGPFKPDHYRY SEQ ID NO:4: MSDKLPYKVADIGLAAWGRKALDIAENEMPGLMRMRERYSASKPLKGARIAGCLHMTVQTAVLIETLVTLGAEVQWSSCNIFSTQDHAAAAIAKAGIPVYAWKGETDE EYLWCIEQTLYFKDGPLNMILDDGGDLTNLIHTKYPQLLPGIRGISEETTTGVHNLYKMMANGILKVPAINVNDSVTKSKFDNLYGCRESLIDGIKRATDVMIAGKVA VVAGYGDVGKGCAQALRGFGARVIITEIDPINALQAAMEGYEVTTMDEACQEGNIFVTTTGCIDIILGRHFEQMKDDAIVCNIGHFDVEIDVKWLNENAVEKVNIKPQ VDRYRLKNGRRIILLAEGRLVNLGCATGHPSFVMSNSFTNQVMAQIELWTHPDKYPVGVHFLPKKLDEAVAEAHLGKLNVKLTKLTEKQAQYLGMSCDGPFKPDHYRY

[0034] In one specific embodiment, the HsSAHH enzyme variant comprises a protein having more than 70% homology to any one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and having HsSAHH enzyme activity.

[0035] In one specific embodiment, the HsSAHH enzyme variant comprises a protein having more than 80%, preferably more than 85%, more preferably more than 95%, and even more preferably more than 99% homology with any one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and having HsSAHH enzyme activity.

[0036] In another typical embodiment, a nucleic acid is provided that encodes the HsSAHH enzyme variant described in any of the preceding embodiments. This nucleic acid sequence can be used for recombinant expression to prepare the aforementioned enzyme variant.

[0037] In another typical embodiment, an expression vector is provided that contains the aforementioned nucleic acid. This expression vector can achieve efficient expression of the aforementioned enzyme variant in prokaryotic or eukaryotic expression systems.

[0038] In another typical embodiment, the application of the above-mentioned HsSAHH enzyme variant in a homocysteine ​​detection reagent is provided. This variant can be used as a key enzyme component in constructing an in vitro enzymatic detection system for homocysteine.

[0039] The term "homocysteine ​​detection reagent" refers to a composition or preparation used for the quantitative detection of homocysteine ​​concentration in biological samples. The term "in vitro enzymatic detection system" refers to a technical platform that uses enzyme-catalyzed reactions as its core to quantitatively analyze target analytes in vitro.

[0040] The principle of homocysteine ​​enzyme cycle detection is as follows: In the presence of S-adenosylmethionine (SAM), homocysteine ​​(Hcy) in the sample is catalyzed by S-adenosylhomocysteine ​​hydrolase (SAHH) to generate S-adenosylhomocysteine ​​(SAH) and methionine. SAH is further hydrolyzed by SAHH to generate Hcy and adenosine. The newly generated Hcy re-enters the above reaction cycle, thereby amplifying the signal. By detecting the adenosine generated, the NADH consumed, or other measurable signals produced during the reaction, the initial concentration of homocysteine ​​in the sample can be quantitatively calculated.

[0041] The commonly used detection method in this field is to quantify nicotinamide adenine dinucleotide (NADH) by continuously monitoring the rate of decrease in absorbance at 340 nm. In the reaction system, Hcy in the sample is converted to S-adenosyl homocysteine ​​(SAH) by S-adenosylmethionine (SAM) and Hcy methyltransferase (HMT). SAH is then hydrolyzed by HsSAHH to regenerate Hcy and adenosine. The newly generated Hcy re-enters the cycle, thus amplifying the signal. Adenosine is catalyzed by adenosine deaminase (ADA) to generate inosine and ammonia (NH3). NH3 reacts with α-ketoglutarate and NADH under the catalysis of glutamate dehydrogenase (GLDH) to generate glutamate and NAD⁺. Since NADH has a characteristic absorption peak at 340 nm while NAD⁺ does not, NADH is continuously consumed as the reaction proceeds, and the absorbance at 340 nm decreases. The absorbance at nm continuously decreases, and the rate of decrease is directly proportional to the initial concentration of Hcy in the sample. The concentration of Hcy in the sample can be quantitatively calculated by measuring the rate of decrease of absorbance and substituting it into the standard curve.

[0042] In another typical embodiment, a homocysteine ​​detection kit is provided, comprising any of the above-mentioned HsSAHH enzyme variants. This kit can be used for the rapid and accurate detection of homocysteine ​​concentration in biological samples. Preferably, the kit further comprises S-adenosylmethionine as a reaction substrate. S-adenosylmethionine reacts with homocysteine ​​under SAHH catalysis to generate S-adenosylhomocysteine, and quantitative analysis of homocysteine ​​is achieved by detecting the reaction product or accompanying signal changes.

[0043] In one specific implementation, the biological sample is selected from one or more of blood, urine, cerebrospinal fluid, and dried blood spots.

[0044] In one specific embodiment, the enzyme activity of wild-type HsSAHH and its variants is determined spectrophotometrically. The core principle is that HsSAHH catalyzes the hydrolysis of the substrate SAH to homocysteine. The free sulfhydryl groups in the generated homocysteine ​​undergo a quantitative colorimetric reaction with a disulfide-containing colorimetric reagent (such as 5,5'-dithiobis(2-nitrobenzoic acid)), generating a product with a characteristic absorption peak in the visible light region. The amount of homocysteine ​​generated can be reflected by measuring the absorbance at this wavelength. A series of standard solutions are prepared using homocysteine ​​standards of known concentrations, and the absorbance is measured under the same colorimetric conditions to plot a concentration-absorbance standard curve. After removing proteins from the enzyme reaction solution using an appropriate method, it is reacted with the colorimetric reagent, and the absorbance is measured and substituted into the standard curve to calculate the amount of homocysteine ​​generated in the enzyme reaction solution. Under the above measurement conditions, the amount of enzyme required to generate one unit amount of homocysteine ​​per unit time is determined. The specific activity of HsSAHH wild-type and its variants was obtained by calculating the enzyme activity per unit mass of protein.

[0045] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0046] Example 1: Expression, purification, and expression level verification of wild-type and mutant HsSAHH

[0047] 1.1 Materials and Reagents

[0048] The gene encoding HsSAHH was synthesized by Genscript Biotech Ltd. (Nanjing, China) and cloned into the pet-28a(+) vector using T4 DNA ligase at the cloning sites of BamHI and XhoI.

[0049] Restriction endonucleases (BamHI, XhoI), FastPfu DNA polymerase, and T4 DNA ligase were purchased from Cypro Biotech (Beijing) Co., Ltd., Tiangen Biotech (Beijing) Co., Ltd., and Takara Bio Engineering (Dalian) Co., Ltd., respectively.

[0050] Escherichia coli DH5α and Rosetta (DE3) competent cells were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0051] LB liquid medium composition: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. TB medium composition: tryptone 12 g / L, yeast extract 24 g / L, glycerol 0.4% (v / v), potassium dihydrogen phosphate 0.017 mol / L, dipotassium hydrogen phosphate 0.072 mol / L.

[0052] 1.2 Construction of the Mutagenesis Library The pet-28a(+) plasmid carrying the wild-type HsSAHH gene was used as the template for all mutagenesis experiments. Site-directed mutagenesis was performed using overlap extension PCR to construct the mutant E59Q variant, the M351T variant, and the double mutant E59Q / M351T variant. All constructed mutants were validated by DNA sequencing. The primer sequences used are shown in Table 1.

[0053] Table 1:

[0054] 1.3 Protein Expression and Purification

[0055] Wild-type and mutant HsSAHH sequences were transformed into Rosetta (DE3) competent cells for expression. Seed cultures were cultured overnight at 37°C with shaking in LB medium containing 50 μg / mL kanamycin. Cultures were then transferred to TB medium containing the corresponding antibiotics and cultured at 37°C for expansion. When the 600 nm optical density (OD600) reached 0.8–1.0, 0.5 mmol / L isopropyl-β-D-thiogalactoside was added to induce protein expression, followed by culture at 18°C ​​and 230 rpm for 16–20 hours with shaking.

[0056] After induction, bacterial cells were collected by centrifugation and resuspended in Tris-HCl buffer (20 mmol / L, pH 8.0) containing 1 mmol / L TCEP-HCl and 10% (w / v) glycerol. Cells were lysed using a high-pressure homogenizer, and cell debris was removed by centrifugation. The supernatant was purified by nickel-iminodiacetic acid (Ni-NTA) affinity chromatography. The clarified lysate was loaded onto a HisTrap column pre-equilibrated with 50 mmol / L nickel sulfate, and washed sequentially with low-concentration imidazole (10–50 mmol / L) and eluted with high-concentration imidazole (200–300 mmol / L) to remove the target protein. The eluent was desalted using a PD-10 column (Cytiva Europe GmbH, Freiburg, Germany) and stored in dialysis buffer. Protein concentration was determined using the Bradford method, with bovine serum albumin as a standard.

[0057] 1.4 Expression and purification results

[0058] SDS-PAGE analysis showed that the wild-type and all mutants were purified to homogeneity, and each protein migrated as a single band with an apparent molecular weight of approximately 48 kDa, consistent with the wild-type enzyme.

[0059] The results of the soluble protein expression level determination are shown in Table 2.

[0060] Table 2:

[0061] The results showed that the expression yield of all three enzyme variants was increased, with the E59Q variant and the M351T variant yielding 1.68 times and 1.86 times that of the wild type, respectively; the double mutant E59Q / M351T variant showed a significantly increased yield, reaching 352 mg / L, which was 3.01 times that of the wild type.

[0062] Example 2: Method for determining HsSAHH enzyme activity and results of activity verification

[0063] 2.1 Reagents and Materials Homocysteine ​​(Hcy), adenosine, S-adenosine homocysteine ​​(SAH), and kanamycin sulfate were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and all were of the highest purity grade.

[0064] 2.2 Measurement Principle HsSAHH enzyme activity was determined spectrophotometrically by quantitatively detecting the amount of homocysteine ​​(Hcy) containing free thiol groups generated in the reaction. The principle is that Hcy reacts with 5,5'-dithiobis(2-nitrobenzoic acid) and DTNB (Ellmann's reagent) to generate a colored product, 2-nitro-5-thiobenzoate (NTB²⁻), which has a characteristic absorption peak at 412 nm.

[0065] Construction of the standard curve: A series of homocysteine ​​standard solutions with concentrations ranging from 1 to 200 μmol / L were prepared using 10 mmol / L phosphate buffer. 200 μL of each standard solution was taken, and 100 μL of 4 mmol / L LTNB was added. After mixing, the solutions were incubated at 37°C in the dark for 30 minutes, followed by measurement of the absorbance at 412 nm. A standard curve was plotted with Hcy concentration on the x-axis and absorbance on the y-axis.

[0066] 2.3 Determination Method

[0067] The purified HsSAHH wild-type or mutant strain, with a final concentration of 1 mg / mL, was mixed with 1 mmol / L of substrate SAH and reacted at 37°C for 1 hour. After the reaction, the protein was removed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. 200 μL of the filtrate was taken, and 100 μL of 4 mmol / L DTNB was added. The absorbance at 412 nm was measured under the same conditions as the standard curve (incubation at 37°C in the dark for 30 minutes). The concentration of Hcy generated was calculated by substituting the absorbance into the standard curve.

[0068] Enzyme activity unit definition: Under the above test conditions, the amount of enzyme required to generate 1 μmol Hcy per minute is defined as one enzyme activity unit (U).

[0069] The specific activity of wild-type and each mutant was determined according to the method described above. All experimental measurements were performed using at least three independent biological replicates. Data are expressed as mean ± standard deviation. Results are shown in Table 3.

[0070] Table 3. Specific activity of wild type and mutant HsSAHH

[0071] The results showed that the specific activities of all enzyme variants were increased, with the E59Q variant and the M351T variant having specific activities that were 1.59 times and 1.41 times that of the wild type, respectively; the double mutant E59Q / M351T variant had the highest specific activity, reaching 2.26 times that of the wild type.

[0072] Example 3. Measurement and Results of Kinetic Parameters

[0073] The kinetic constants (Michaelis constant, Km; turnover number, kcat; catalytic efficiency, kcat / Km) were determined by measuring the initial reaction rate at different SAH concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM). The reaction was carried out at 37°C in 50 mM phosphate-buffered saline (pH 7.5) containing 1 KU of purified wild-type and mutant enzymes. The MyCurveFit online tool was used. https: / / mycurvefit.com / These parameters were calculated by fitting the data to the classical Michaelis equation. The results are shown in Table 4.

[0074] Table 4. Kinetic parameters of wild-type and mutant HsSAHH

[0075] The results showed that the catalytic efficiency of the enzyme variants was improved. The catalytic efficiency of the E59Q variant and the M351T variant was 2.91 times and 2.24 times that of the wild type, respectively. The catalytic efficiency of the double mutant E59Q / M351T variant reached 3.93 times that of the wild type.

[0076] Molecular dynamics simulations were performed to investigate the structural mechanisms underlying the enhanced catalytic performance. All simulations were conducted using the GPU-accelerated GROMACS 2024.3 software package. Protein residues were modeled using the AMBER14SB force field, with missing hydrogen atoms added accordingly. SAH ligands were parameterized using the generalized AMBER force field (GAFF) via the ACPYPE online server (https: / / bio2byte.be / acpype / ). The system was solvated with TIP3P water molecules in a periodic cubic cell, with solute atoms maintained at least 1 nm away from the cell boundary. The protonation state was adjusted to achieve system electroneutrality, and counterions (Na+ and Cl-) were added as needed. Energy minimization was performed using a 50,000-step steepest descent algorithm to eliminate unfavorable atomic contacts. Subsequently, the protein-ligand complex was equilibrated using a two-step procedure: first, a 2 ns NVT simulation was performed to heat the system from 0 to 310 K, followed by a 2 ns NPT simulation at 310 K to stabilize the pressure. Throughout the equilibrium process, hydrogen bonds were constrained using the Linear Constraint Solver (LINCS) algorithm, and long-range electrostatic interactions were handled using the Particle Mesh Ewald (PME) method with a cutoff distance of 1.0 nm. After equilibrium was achieved, three independent 300 ns production runs of the complete system were performed at 310 K, each using different atomic velocities to improve sampling statistics. The integration time step was 5 fs, and trajectory frames were saved every 100 ps for subsequent analysis.

[0077] To investigate the structural basis for the maintenance of thermal stability, molecular dynamics simulations were performed at 310 K for 300 ns. Figure 1 As shown, root mean square deviation (RMSD) analysis reveals that the E59Q / M351T variant exhibits considerable structural stability compared to the wild-type HsSAHH. Both systems exhibited significant conformational fluctuations in the initial stages of the simulation, subsequently reaching a stable equilibrium state after approximately 200 ns and maintaining structural consistency for the remainder of the trajectory. Therefore, all subsequent analyses were performed using the equilibrium trajectory frames (200–300 ns).

[0078] 4.1 Substrate binding affinity Enzyme-substrate binding affinity was quantified using the MM / PBSA method. Based on the RMSD convergence curve (… Figure 1The trajectories (200–300 ns) of the wild-type and mutant enzymes after equilibrium were extracted for energy calculation. The total binding free energy was decomposed into four components: van der Waals interaction energy (ΔE_VDW), electrostatic interaction energy (ΔE_EL), polar solvation energy (ΔE_GB), and nonpolar solvation energy (ΔE_SURF). Under the dynamic equilibrium assumption, the entropy contribution was considered negligible; therefore, ΔG_bind is thermodynamically equivalent to the total free energy.

[0079] Table 5. MM-GBSA contribution decomposition analysis of wild-type and mutant HsSAHH–SAH complexes

[0080] Free energy calculations showed that the E59Q / M351T variant had a significantly more favorable ΔG_bind (–57.20 ± 2.34 kcal / mol) compared to the wild-type enzyme (–34.81 ± 1.63 kcal / mol). This enhanced affinity stemmed from optimized contributions from multiple energy components. Electrostatic interactions were the primary driving force for substrate binding in both systems, as evidenced by their dominant and favorable contribution. Van der Waals interactions also significantly stabilized the enzyme-substrate complex, but to a lesser extent than the electrostatic effect. Nonpolar solvation energy provided a modest but favorable contribution, indicating the presence of a stable hydrophobic effect.

[0081] Quantitative comparisons of the energy terms confirmed the enhanced binding capacity of the mutant: van der Waals interactions increased from –43.71 ± 2.44 kcal / mol in the wild type to –45.28 ± 0.02 kcal / mol in the mutant; electrostatic interactions improved from –124.35 ± 5.75 kcal / mol to –135.80 ± 0.18 kcal / mol; and nonpolar solvation energy increased from –6.04 ± 0.13 kcal / mol to –6.67 ± 0.25 kcal / mol. Therefore, the improved energy distribution promotes more efficient molecular recognition and transition state stabilization, thereby contributing to an increased catalytic rate of the E59Q / M351T variant.

[0082] 4.2 Substrate channels and catalytic cavities The substrate entry pathways of wild-type and mutant HsSAHH were computationally analyzed using CAVER Web v1.2.

[0083] Table 6. Channel information for wild type and HsSAHH variant

[0084] As summarized in Table 6, the E59Q / M351T variant exhibits a significantly expanded substrate channel with a bottleneck radius of 1.7 Å, compared to 1.0 Å for the wild-type enzyme. Simultaneously, the channel length decreased from 12.1 Å to 10.1 Å. Furthermore, the channel structure underwent remodeling, with the curvature increasing from 0.64 in the wild-type to 0.75 in the mutant, indicating a conformational change in the substrate entry pathway.

[0085] Further use of Prank Web server ( https: / / prankweb.cz / The active site cavity was further analyzed.

[0086] Table 7. Cavity volume of wild type and HsSAHH variant

[0087] The results showed that the estimated cavity volume of the E59Q / M351T variant increased to 3645.2 ų, while that of the wild-type HsSAHH was 3314.6 ų. It is well known that channel geometry and the configuration of the catalytic cavity have a significant impact on enzyme catalytic efficiency and substrate selectivity. Therefore, an enlarged active site cavity, a wider channel bottleneck, and a shorter channel length help facilitate efficient substrate entry and optimize solvent localization.

[0088] 4.3 Pronuclear attack range

[0089] The hydrolysis of SAH is initiated by a nucleophilic attack on the 3′-hydroxyl group of the substrate ribose ring by the Nζ atom of K186. The closer the catalytic nucleophile is to the reaction center of the substrate, the more efficient the catalytic attack.

[0090] according to Figure 2 As shown, in the E59Q / M351T variant–SAH complex, the distance between K186-Nζ and SAH-3′-OH was measured to be 3.1 Å ( Figure 2 a), significantly shorter than the corresponding value of 4.6 Å observed in the wild-type complex ( Figure 2 (b) This significantly shortened 1.5 Å distance places the nucleophile in a catalytically optimal arrangement, providing a structural explanation for the enhanced catalytic activity of the E59Q / M351T mutant compared to the wild type.

Claims

1. An HsSAHH enzyme variant, characterized in that, Compared to the wild-type HsSAHH shown in SEQ ID NO: 1, this variant contains one or more amino acid modifications in the metal coordination motif of the wild-type HsSAHH.

2. The variant according to claim 1, characterized in that, The metal coordination motif includes a first metal coordination motif and a second metal coordination motif; the first metal coordination motif is amino acids at positions 55-59; and the second metal coordination motif is amino acids at positions 351-355.

3. The variant according to claim 2, characterized in that, The amino acid modification includes a first amino acid modification and / or a second amino acid modification; the first amino acid modification is to mutate the 55th amino acid in the first metal coordination motif, and the second amino acid modification is to mutate the 351st amino acid in the second metal coordination motif.

4. The variant according to claim 3, characterized in that, The amino acid modification includes: the first amino acid modification is to mutate the amino acid at position 55 to glutamine; the second amino acid modification is to mutate the amino acid at position 351 to threonine.

5. The variant according to claim 4, characterized in that, The variant is selected from one of the following, 1) E59Q variant; 2) M351T variant; 3) E59Q / M351T variant.

6. A nucleic acid, characterized in that, The nucleic acid encodes a variant as described in any one of claims 1-5.

7. An expression carrier, characterized in that, The vector comprises the nucleic acid as described in claim 6.

8. The application of the HsSAHH enzyme variant in homocysteine ​​detection reagents, characterized in that, The HsSAHH enzyme variant is selected from the variants described in any one of claims 1-4.

9. A homocysteine ​​detection kit, characterized in that, It includes the HsSAHH enzyme variant as described in any one of claims 1-5.

10. The reagent kit according to claim 8, characterized in that, The kit also includes S-adenosylmethionine.

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