Mono-iron hydrogenase model compound containing 3, 4, 5-substituted pyridine ligand and recombinase thereof
By designing and synthesizing a monoferric hydrogenase model with pyridine ligands substituted at the 3, 4, and 5 positions and recombining it with the apoenzyme, the problem of low catalytic activity in the existing technology was solved, and an efficient catalytic hydrogen heterolytic reaction was achieved.
Patent Information
- Application Number
- CN202510191129.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-09-16
AI Technical Summary
The catalytic activity of existing artificially synthesized monoferric hydrogenase model substances is low, and there is still a large gap between their recombinant activity and that of natural monoferric hydrogenase.
A class of monoferric hydrogenase model compounds with pyridine ligands substituted at the 3, 4, and 5 positions were designed and synthesized, and recombinant enzymes with higher catalytic activity were formed by recombination with apoenzymes.
The catalytic activity of the recombinant enzyme is improved, so that it exhibits higher oxidation and reduction activity in the catalytic hydrogen heterolytic reaction, approaching or exceeding the level of the existing technology.
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Abstract
Description
Technical Field
[0001] The present invention relates to the fields of coordination chemistry and metal organic chemistry, in particular to a class of monoferric hydrogenase model substances containing pyridine ligands substituted at the 3, 4 and 5 positions and recombinant enzymes thereof. Background Art
[0002] Monoferric hydrogenase is the last of the three hydrogenase classes (nickel-iron hydrogenase, iron-iron hydrogenase, and monoferric hydrogenase) to have its metal active center structure determined. Unlike the other two classes of hydrogenase, its structure lacks an iron-sulfur cluster and can directly catalyze hydrogen into hydride and hydride ions, offering greater potential for its subsequent application in organic chemistry. In the natural monoferric hydrogenase metal center, the iron is coordinated by a didentate hydroxypyridine, two cis-CO groups, a sulfur from cysteine, and an H2O molecule. For the structure of the metal center of monoferric hydrogenase, see: S.Shima, O.Pilak, S.Vogt, M.Schick, MSStagni, W.Meyer-Klaucke, E.Warkentin, RKThauer andU.Ermler, Science 2008,321,572-575; T.Hiromoto, K.Ataka, O.Pilak, S.Vogt, MSStagni, W.Meyer-Klaucke, E.Warkentin, RKThauer, S.Shima and U.Ermler, FEBS Lett.2009,583,585-590.
[0003] Since the structure of the metal active center of monoferric hydrogenase was determined, scientists have synthesized a large number of monoferric hydrogenase models with different structures. Among them, the research groups of Professor Hu Xile at the Swiss Federal Institute of Technology in Lausanne (EPFL), Academician Song Licheng at Nankai University, and Professor Michael J. Rose at the University of Texas at Austin (UT Austin) have conducted extensive research in this field and published a series of research works. See: T. Xu, D. Chen and X. Hu, Coordin. Chem. Rev. 2015, 303, 32-41; TAManes and MJ Rose, Coordin. MJ Rose, Curr. Opin. Chem. Biol. 2022, 66, 102096; C. Wang, Z. Lai, G. Huang and H.-J. Pan, Chem. Eur. J. 2022, 28, e202201499.
[0004] However, until now only a few monoferric hydrogenase model compounds have catalytic activity. In 2014, Professor Franc Meyer and others first realized the splitting of hydrogen and hydrogenation of substrates by ruthenium model compounds. Professor Hu Xile's research group reported iron model compounds and manganese model compounds with catalytic hydrogenation activity in 2016 and 2019, respectively. Professor Michael J Rose's research group also reported several monoferric hydrogenase model compounds with catalytic activity between 2017 and 2019. However, most of the above work was carried out in organic solutions and the reaction efficiency was low. In addition, these models have not been recombined with proteins, nor have they completed the conversion reaction of natural substrates. See: KF Kalz, A. Brinkmeier, S. Dechert, R. A. Mata and F. Meyer, J. Am. Chem. Soc. 2014, 136, 16626-16634; T. Xu, C.-J. M Yin, MD Wodrich, S. Mazza, KM Schultz, R. Scopeliti and X.Hu,J.Am.Chem.Soc.2016,138,3270-3273; J.Seo,TAManes,MJRose,Nat.Chem.2017,9,552–557;SAKerns,A.-C.Magtaan,PR Vong, MJ Rose, Angew. Chem. Int. Ed. 2018, 57, 2855–2858; YI Cho, G. Durgaprasad, MJ Rose, Inorg. Chem. 2019, 58, 12689–12699.
[0005] In 2015, the Seigo Shima group at the Max Planck Institute in Germany and the Professor Hu Xile group at the Swiss Federal Institute of Technology in Lausanne reported the first artificial recombinant of a single iron hydrogenase model with an iron hydrogenase apoenzyme, and measured its oxidation and reduction activity. After that, they reported the use of a manganese model and the recombinant iron hydrogenase apoenzyme in 2019 and 2021, and obtained better catalytic activity. However, even though these artificially synthesized models have certain catalytic activity, there is still a big gap compared to the activity after recombinant monoiron hydrogenase natural metal center (FeGP). Therefore, it is very important to synthesize some single iron hydrogenase model compounds with higher catalytic activity. See: S. Shima, D. Chen, T. Xu, MD Wodrich, T. Fujishiro, K M Schultz, J. Kahnt, K. Ataka and X.Hu,Nat.Chem.2019,11,669-675;H.-J.Pan,G.Huang,MDWodrich,FFTirani,K.Ataka,S.Shima and X.Hu,Angew.Chem.Int.Ed.2021,60,13350-13357. Summary of the Invention
[0006] To address the low activity of synthetic monoferric hydrogenase models, the present invention provides a class of monoferric hydrogenase models with pyridine ligands substituted at the 3, 4, and 5 positions. This model has simple synthesis steps, high yield, and stable structure. The recombinant enzyme exhibits high catalytic activity after reconstitution with an apoenzyme.
[0007] The specific technical solutions of the present invention are as follows:
[0008] A type of monoferrohydrogenase model, the chemical structure of which is shown below:
[0009] where R 1 、R 2 are the same or different monodentate ligands or R 1 、R 2 Represents a bidentate ligand. R 3 、R 4 R are the same or different and represent H or a C1-C6 alkyl group. 4 Represents H or hydroxyl. The recombinant enzyme obtained by recombining the monoferric hydrogenase model substance with the apoenzyme has good stability and high catalytic activity.
[0010] Preferably, the monodentate ligand is selected from NH3, F - 、Cl - Br - , I - 、S 2- 、SCN - 、NO 3- 、N3 - , acetonitrile, pyridine, triphenylphosphine, carbon monoxide CO, the bidentate ligand is selected from ethylenediamine, oxalate, nitrite, 2-mercaptoethanol, 2-mercaptopyridine, 6-methyl-2-mercaptopyridine, and acetate.
[0011] Preferably, the R 1 、R 2 The same or different ligands are selected from halogen, or are bidentate ligands.
[0012] Preferably, the R 3 、R 4 are the same or different and represent H or a C1-C6 alkyl group.
[0013] Preferably, the R 5 represents H or hydroxyl.
[0014] More preferably, the R 1 、R 2 Same or different, represents Cl - Br - or I - R 3 、R 4 Same or different, represents methyl or ethyl, R 5 Represents hydroxyl.
[0015] In a specific example of the present invention, the R 1 、R 2 Represents Cl - , R 3 、R 5 Represents methyl, R 4 Represents hydroxyl.
[0016] The monoferric hydrogenase model of the present invention can form a salt with a cation. The cation is selected from NH4 + 、Na + , K + 、Ag + , tetrabutylammonium or tetraethylammonium. Preferably, the cation is selected from tetrabutylammonium.
[0017] Another object of the present invention is to provide a recombinant monoferric hydrogenase, which is recombined by the monoferric hydrogenase model of the present invention and an apoenzyme (natural monoferric hydrogenase with the active center removed).
[0018] The natural monoferric hydrogenase is selected from one or more monoferric hydrogenases derived from (M.ja) Methanococcus jannaschii, (M.ig) Methanotorris igneus, (M.in) Methanocaldococcus infernus, (M.ae) Methanococcusaeolicus, (M.pa) Methanolacinia paynteri, (M.fo) Methanoregula formicica.
[0019] In a specific example of the present invention, the natural monoferric hydrogenase is a monoferric hydrogenase derived from (M.ja) Methanococcusjannaschii.
[0020] Another object of the present invention is to provide the use of the recombinant monoferric hydrogenase in catalyzing hydrogen heterolytic cleavage reactions. The monoferric hydrogenase model is recombined with an apoenzyme to form a recombinant monoferric hydrogenase with catalytic activity, catalyzing hydrogen into negative hydrogen ions and positive hydrogen ions.
[0021] In a specific example of the present invention, the recombinant monoferric hydrogenase catalyzes the oxidation reaction of methylene-H4MPT or catalyzes the oxidized state of methenyl-H4MPT. + hydrogenation reaction.
[0022] The present invention also provides a method for preparing the monoferric hydrogenation model of the 3, 4, 5-substituted pyridine ligand, using R 1 、R 2 Represents Cl, R 3 、R 5 Represents methyl, R 4 Taking hydroxyl group as an example, the method comprises the following steps:
[0023]
[0024]
[0025] 1) Method 1: Under nitrogen protection, Na2Fe(CO)4·(1,4-dioxane) 1.5 The THF solution was placed at -50°C, followed by the addition of the pyridine ligand. The reaction was continued for 30 minutes, then the temperature was lowered to -20°C for another 30 minutes. Finally, the temperature was lowered to -60°C, I was added, and the reaction was continued for 1 hour. The resulting reaction solution was brought to room temperature, and n-tetrabutylammonium fluoride was added. The reaction was continued at room temperature for 10 minutes, and the solvent was removed to obtain a black solid.
[0026] Method 2: Under nitrogen, place the THF solution of the pyridine ligand in an ice bath. Then, inject n-butyl lithium. After reacting for 30 minutes, remove the mixture to -50°C, inject a tetrahydrofuran solution of Fe(CO)₅, and slowly warm it to -20°C. Then, cool it to -60°C, add I₂, and continue the reaction for 1 hour. Bring the resulting reaction mixture to room temperature, add n-tetrabutylammonium fluoride, and react at room temperature for 10 minutes. Remove the solvent to obtain a black solid.
[0027] 2) Using dichloromethane and methanol as developing solvents, the black solid obtained in step 1) was purified by column chromatography, and the red main band was collected, which was the monoferrohydrogenase model precursor A with methyl groups at positions 3 and 5, hydroxyl groups at positions 4, and methoxy groups at positions 2 of pyridine. Its structure is shown below:
[0028]
[0029] 3) The monoferrohydrogenase model precursor A obtained in step 2) above was dissolved in dichloromethane, and n-tetrabutylammonium bromide was added. The mixture was placed at 0° C., and aluminum tribromide was added. The mixture was reacted for 2 h and then quenched with 1 M dilute hydrochloric acid.
[0030] 4) The organic layer in step 3) was dried under reduced pressure and separated by column chromatography using dichloromethane and methanol as developing solvents. The red main band was collected, which was the monoferric hydrogenase model compound 1 with methyl groups at the 3 and 5 positions of pyridine and hydroxy groups at the 2 and 4 positions. Its structure is shown below:
[0031]
[0032] Compared with existing artificially synthesized model substances and naturally extracted monoferric hydrogenase metal catalytic centers, the advantages of the present invention are: 1) The monoferric hydrogenase model substance of the present invention is more similar in structure to the natural catalytic center, and the catalytic activity of the recombinant enzyme after recombinant treatment is higher than the recombinant enzyme activity of the monoferric hydrogenase model substance reported in the prior art.
[0033] 2) The monoferrohydrogenase model of the present invention is simple to synthesize, has a high yield, can produce gram-level products, has a stable structure, and meets the requirements of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 . Monoferrohydrogenase model precursor A 1 H NMR spectrum.
[0035] Figure 2 .Single crystal structure of precursor A of the monoferrohydrogenase model.
[0036] Figure 3 . Monoferrohydrogenase model 1 1 H NMR spectrum.
[0037] Figure 4. Monoferrohydrogenase model 1 13 C NMR spectrum.
[0038] Figure 5 .Infrared spectrum of monoferrohydrogenase model 1.
[0039] Figure 6 .Single crystal structure of monoferrohydrogenase model 1.
[0040] Figure 7 .High-resolution mass spectrum of monoferrohydrogenase model 1 (negative mode).
[0041] Figure 8 .Single crystal structure of the monoferrohydrogenase model 1-iodine (the two halogen ligands are iodide ions and the positive ion is tetrabutylammonium).
[0042] Figure 9 .Single crystal structure of the monoferric hydrogenase model 1-bromo (the two halogen ligands are bromide ions and the positive ion is tetrabutylammonium).
[0043] Figure 10 . Monoferrohydrogenase model 2 1 H NMR spectrum.
[0044] Figure 11 . Monoferrohydrogenase model 2 13 C NMR spectrum.
[0045] Figure 12 .Infrared spectrum of monoferrohydrogenase model 2.
[0046] Figure 13 .High-resolution mass spectrum of monoferrohydrogenase model 2 (negative mode).
[0047] Figure 14 . Monoferrohydrogenase model 3 1 H NMR spectrum.
[0048] Figure 15 . Monoferrohydrogenase model 3 13 C NMR spectrum.
[0049] Figure 16 .Infrared spectrum of monoferrohydrogenase model 3.
[0050] Figure 17 .High-resolution mass spectrum of monoferrohydrogenase model 3 (negative mode).
[0051] Figure 18 . Monoferrohydrogenase model 4 1 H NMR spectrum.
[0052] Figure 19. Monoferrohydrogenase model 4 13 C NMR spectrum.
[0053] Figure 20 .Single crystal structure of the monoferric hydrogenase model 3-bromo-chloro (the two halogens are bromide ion and chloride ion, and the positive ion is tetrabutylammonium).
[0054] Figure 21 .High-resolution mass spectrum of monoferrohydrogenase model 5 (containing 2-mercaptoethanol ligand, negative mode).
[0055] Figure 22 .High-resolution mass spectrum of monoferrohydrogenase model 6 (containing acetate ligand, negative mode).
[0056] Figure 23 .High-resolution mass spectrum of monoferric hydrogenase model 7 (containing 2-mercaptoethanol ligand, negative mode).
[0057] Figure 24 .High-resolution mass spectrum of monoferric hydrogenase model 8 (containing 2-mercaptoethanol ligand, negative mode).
[0058] Figure 25 .Absorption change diagram at 336nm during substrate oxidation catalyzed by the recombinant enzyme 1 prepared in Example 4.
[0059] Figure 26 .Absorption change diagram at 336nm during the substrate hydrogenation catalyzed by the recombinant enzyme 1 prepared in Example 4. DETAILED DESCRIPTION
[0060] In order to better understand the present invention, the solutions of the present invention will be further described below through specific examples, but the protection scope of the present invention should include all the contents of the claims and is not limited thereto.
[0061] Example 1: Preparation of Monoferrohydrogenase Model Precursor
[0062]
[0063] Method 1:
[0064] 1) Under nitrogen protection, 346 mg (1 mmol) Na2Fe(CO)4·(1,4-dioxane) 1.5 A THF solution (15 mL) of 4-silyl-protected pyridine ligand was placed at -50°C, followed by the addition of 315 mg (1 mmol) of a 4-silyl-protected pyridine ligand. The reaction was allowed to proceed for 30 minutes, then the temperature was moved to -20°C for another 30 minutes. Finally, the temperature was lowered to -60°C, 253.8 mg (1 mmol) of I2 was added, and the reaction was continued for 1 hour.
[0065] 2) The reaction solution was brought to room temperature, and a tetrahydrofuran solution of n-tetrabutylammonium fluoride (1.2 mmol) was added. The mixture was reacted at room temperature for 10 min and then dried to obtain a black solid.
[0066] 3) The black solid obtained above was dissolved in dichloromethane, and column chromatography was performed using dichloromethane and methanol (volume ratio 20:1) as a developing solvent to collect the red main band, and the solvent was removed under reduced pressure to obtain the monoferric hydrogenase model precursor A245.
[0067] mg, yield: 53.2%.
[0068] Method 2:
[0069] 1) Under nitrogen, place a THF solution of 281 mg (1 mmol) of a 4-silyl-protected pyridine ligand in an ice bath. Then, inject 0.48 mL (1.2 mmol, 2.5 M in hexane) of n-butyl lithium. After reacting for 30 minutes, move the mixture to -50°C and inject 0.12 mL (1.1 mmol) of a tetrahydrofuran solution of Fe(CO)5. The temperature is slowly raised to -20°C, then cooled to -60°C, and I2 is added. The reaction is continued for 1 hour.
[0070] 2) The resulting reaction solution was brought to room temperature, and n-tetrabutylammonium fluoride was added. The reaction was carried out at room temperature for 10 minutes, and then the solvent was removed to obtain a black solid.
[0071] The black solid obtained above was dissolved in dichloromethane and separated by column chromatography using dichloromethane and methanol (volume ratio 20:1) as the developing solvent. The red main band was collected and the solvent was removed under reduced pressure to obtain 258 mg of monoferrohydrogenase model precursor A, with a yield of 56.1%. The structural data of monoferrohydrogenase model precursor A are as follows: IR (soild): v C＝O 2082(s),2029(s),2003(s),1651(s)cm -1.1 H NMR (400MHz, Acetone) δ9.48 (s, 1H), 4.75 (d, J = 20.9Hz, 1H), 4.27 (d, J = 20.8Hz, 1H), 4.05 (s, 3H), 2.30 (s, 3H), 2.21 (s, 3H). 13 C NMR(151MHz,Acetone)δ256.86(s),210.46(s),209.69(s),200.05(s),164.73(s),16 4.38(s),156.25(s),115.99(s),111.73(s),66.16(s),60.64(s),11.83(s),9.56(s).
[0072] Monoferrohydrogenase model precursor A 1 H NMR spectrum Figure 1 The single crystal structure is shown in Figure 2 shown.
[0073] Example 2: Preparation of Monoferric Hydrogenase Models 1, 1-Bromo, 1-Iodine, and 1-Bromo-Chlorine Containing 2,4-Dihydroxy-3,5-Dimethylpyridine Ligands
[0074]
[0075] 1) Under nitrogen protection, 460 mg (1 mmol) of model precursor A was mixed with 225.7 mg (0.7 mmol) of n-tetrabutylammonium bromide, dissolved in dichloromethane, and placed in an ice bath.
[0076] 2) Aluminum tribromide (931 mg, 3.5 mmol) was added to the above solution, and the mixture was reacted for 2 h and then quenched with 1 M dilute hydrochloric acid.
[0077] 3) The organic layer was dried under reduced pressure to obtain a black solid.
[0078] 4) The black solid obtained above was dissolved in dichloromethane and separated by column chromatography using dichloromethane and methanol (volume ratio 10:1) as a developing solvent. The main red band was collected and the solvent was removed under reduced pressure to obtain 241 mg of the monoferrohydrogenase model compound 1 (yield: 31.4%).
[0079] The structural data of the monoferrohydrogenase model 1 are as follows: IR (soild): v C＝O 2032(s),1958(s),1646(s)cm -1.1 HNMR (400MHz, Acetone) δ12.52(s,1H),8.57(s,1H),4.29(d,J=20.4Hz,1H),3.54(d,J=19.9Hz,1H),3.51–3.3 0(m,8H),2.08(s,3H),2.06(s,3H),1.79(dd,J=8.3,4.5Hz,8H),1.41(q,J=7.4Hz,8H),0.96(t,J=7.3Hz,12H). 13 C NMR(151MHz,Acetone)δ263.20(s),211.96(s),211.15(s),165.27(s),162.32(s),153.54(s),1 10.28(s),103.04(s),60.37(s),59.48(s),24.25(s),20.10(s),13.51(s),11.23(s),8.34(s).
[0080] Monoferrohydrogenase model 1 1 H NMR spectrum Figure 3 shown. 13 C NMR spectrum Figure 4 The infrared spectrum is shown as Figure 5 The single crystal structure is shown in Figure 6 As shown. High resolution mass spectrum (negative mode) is as follows Figure 7 shown.
[0081] 5) In step 2), if 1M hydrobromic acid or 1M hydroiodic acid is used for quenching, the monoferric hydrogenase model compounds 1-bromo and 1-iodo can be obtained, respectively, and their structures are as follows:
[0082]
[0083] The single crystal structure of the monoferrohydrogenase model 1-iodine (two halogen ligands are iodide ions and the positive ion is tetrabutylammonium) is shown in the figure below. Figure 8 The single crystal structure of the monoferric hydrogenase model 1-bromo (two halogen ligands are bromide ions and the positive ion is tetrabutylammonium) is shown in Figure 9 shown.
[0084] 6) In step 2), if a mixed solution of hydrobromic acid and hydrochloric acid (1:1) is used for quenching, a monoferric hydrogenase model compound 1-bromo-chloro can be obtained, the structure of which is as follows:
[0085]
[0086] Referring to the above method, other monoferric hydrogenase model compounds were prepared by selecting different 3-, 4-, and 5-substituted pyridine ligands and different halogens:
[0087] Monoferrohydrogenase model 2: IR(soild):v C＝O 2025(s),1952(s),1629(s)cm -1 . 1 H NMR (600MHz, Acetone) δ12.54(s,1H),10.74(s,1H),6.60(s,1H),6.21(s,1H),4.48(dd,J=19.3 Hz,1H),3.52(dd,J=19.4Hz,1H),3.44–3.29(m,23H),1.77(s,23H),1.40(q,23H),0.96(t,35H). 13C NMR(151MHz,Acetone)δ263.47,213.29,212.05,170.45,168.65,160.25,103.45,95 .63,62.72,59.53,24.64,20.52,14.00.HRMS(ESI)calcd.forC6H6Cl2FeNO2[M-3CO] - m / z249.9125, found 249.9130.
[0088] Monoferrohydrogenase model 2 1 H NMR spectrum Figure 10 shown. 13 C NMR spectrum Figure 11 The infrared spectrum is shown as Figure 12 As shown. High resolution mass spectrum (negative mode) is as follows Figure 13 shown.
[0089] Monoferrohydrogenase model 3: IR(soild):v C＝O 2025(s),1954(s),1619(s)cm -1 . 1 H NMR (600MHz, Acetone) δ12.36(s,1H),10.44(s,1H),6.39(s,1H),4.28(dd,J=20.3Hz,1H),3.53(dd,J=20 .1Hz,1H),3.43–3.31(m,14H),1.99(s,3H),1.77(s,14H),1.40(q,J=7.5Hz,14H),0.96(t,J=7.3Hz,21H). 13 C NMR(151MHz,Acetone)δ263.94,213.15,212.15,168.11,166.34,157.66,111.35,95.72,61.29,59.52,24.61,20.50,13.98,11.15.HRMS(ESI)calcd.for C7H8Cl2FeNO2[M-3CO] - m / z 263.9282, found 263.9288.
[0090] Monoferrohydrogenase model 3 1 H NMR spectrum Figure 14 shown. 13 C NMR spectrum Figure 15 The infrared spectrum is shown as Figure 16 As shown. High resolution mass spectrum (negative mode) is as follows Figure 17 shown.
[0091] Monoferrohydrogenase model 4: 1 H NMR (400MHz, Acetone) δ12.74(s,1H),7.38(s,1H),4.31(d,J=20.4Hz,1H),3.57(d,J=20.1 Hz,1H),3.48–3.34(m,8H),2.14(s,6H),1.80(s,8H),1.43(d,J=7.3Hz,8H),0.97(s,12H). 13 C NMR (151MHz, Acetone) δ263.23,212.82,211.53,165.75,154.36,142.74,121.54,119.26,60.77,59.58,24.56,20.43,18.05,15.80,13.92.
[0092] Monoferrohydrogenase model 4 1 H NMR spectrum Figure 18 shown. 13 C NMR spectrum Figure 19 shown.
[0093] Example 3: Replacement of the ligand of the monoferric hydrogenase model 1 containing a 2,4-dihydroxy-3,5-dimethylpyridine ligand
[0094]
[0095] 1) Under nitrogen protection, 160 mg (0.23 mmol) of monoferrohydrogenase model compound 1 was dissolved in 5 ml of dichloromethane.
[0096] 2) 19.9 mg (0.26 mmol) of 2-mercaptoethanol and 25.8 mg (0.26 mmol) of triethylamine were added to the above solution and reacted at room temperature for 30 minutes.
[0097] 3) The above solution was added to 30 ml of icy n-hexane to precipitate a large amount of yellow solid, which was centrifuged and washed with water to obtain the monoferrohydrogenase model compound 5. Yield: 68.7%.
[0098] HRMS(ESI)calcd.for C 13 H 14 FeNO6S[MH] - m / z 367.9891, found 367.9899. High-resolution mass spectrum (containing 2-mercaptoethanol ligand, negative mode) is as follows Figure 21 shown.
[0099] Referring to the above method, different monoferronihydrogenase model compounds and different bidentate ligands were selected to prepare other monoferronihydrogenase model compounds:
[0100] Monoferrohydrogenase model 6: HRMS(ESI)calcd.for C 13 H 12 FeNO7 - [MH] - m / z349.9969,found 349.9969;calcd.for C 10 H 12 FeNO4 - [M-3CO-H] - m / z 266.0121, found 266.0122. High-resolution mass spectrum (with acetate ligand, negative mode) is as follows Figure 22 shown.
[0101] Monoferrohydrogenase model 7: HRMS(ESI)calcd.for C 11 H 10 FeNO6S - [MH] - m / z339.9584,found 339.9583;calcd.for C8H 10 FeNO3S - [M-3CO-H] - m / z 255.9736, found 255.9734. High-resolution mass spectrum (containing 2-mercaptoethanol ligand, negative mode) is as follows Figure 23 shown.
[0102] Monoferrohydrogenase model 8: HRMS(ESI)calcd.for C 12 H 12 FeNO6S - [MH] - m / z353.9740,found 353.9741;calcd.for C9H 12 FeNO3S - [M-3CO-H] - m / z 269.9893, found 269.9893. High-resolution mass spectrum (containing 2-mercaptoethanol ligand, negative mode) is as follows Figure 24 shown.
[0103] Preparation of comparative model:
[0104] The comparative model was prepared according to the method disclosed in the reference (S. Shima, D. Chen, T. Xu, MD Wodrich, T. Fujishiro, KM Schultz, J. Kahnt, K. Ataka and X. Hu, Nat. Chem. 2015, 7, 995-1002).
[0105] Example 4: Recombination of Monoferric Hydrogenase Model with Apoenzyme
[0106] 1) The monoferrohydrogenase model substances 1-4 and the comparative model substance prepared in Example 2 were dissolved in methanol (containing 1% by volume of 2-mercaptoethanol) to a final concentration of 10 mM.
[0107] 2) To a sample vial, add 20 μL of 100 mM guanosine 5′-monophosphate disodium salt aqueous solution, 20 μL of apoenzyme (prepared by the methods disclosed in Shima, S. & Thauer, R. K. in Methods Enzymology Vol. 331 317-353 (Academic Press, 2001) and Shima, S., Schick, M. & Tamura, H. Chapter seven - Preparation of [Fe]-Hydrogenase from Methanogenic Archaea. Methods Enzymology 494, 119-137 (2011)) (40 mg / ml), and 310 μL of pH 5.6 sodium acetate buffer solution (100 mM), respectively, and place on ice.
[0108] 3) 50 μL of the model solution from step 1) was added to the solution from step 2) and incubated on ice for 1 hour to obtain recombinant enzymes 1 to 4 and a comparative recombinant enzyme, which were prepared to a concentration of 2 mg / ml for later use.
[0109] Example 5: Catalytic oxidation activity test of the recombinant enzyme based on the monoferric hydrogenase model on the substrate methylene-H4MPT. The reaction equation is as follows: The oxidation effects of the recombinant enzymes 1 to 4 prepared in Example 4 and the comparative recombinant enzymes on the substrate were investigated. The specific method is as follows:
[0110] 1) Take a quartz cuvette and add 780 μL of 120 mM pH 6.0 potassium phosphate buffer.
[0111] 2) Inject 10 μL of 4.4 mM substrate methylene-H4MPT into the cuvette and measure the absorbance at 336 nm using a UV-visible spectrophotometer.
[0112] Inject 5 μL of recombinant enzyme (2 mg / ml) into the above cuvette, detect the change in absorbance at 336 nm, and calculate its activity. The calculation formula is:
[0113]
[0114] The catalytic activity results of recombinases 1 to 4 and the comparative recombinase are shown in Table 1. The absorption change at 336 nm during substrate oxidation catalyzed by recombinase 1 is shown in Table 1. Figure 25 shown.
[0115] Example 6: Recombinant enzyme based on monoferric hydrogenase model for substrate methenyl-H4MPT + The catalytic hydrogenation activity test reaction equation is as follows:
[0116]
[0117] The hydrogenation effect of the recombinant enzymes 1 to 4 prepared in Example 4 and the comparative recombinant enzyme on the substrate was examined. The specific method was as follows: 1) Take a quartz cuvette and add 780 μL of 120 mM pH 7.5 potassium phosphate buffer.
[0118] 2) Inject 10 μL of 4.4 mM oxidized substrate methenyl-H4MPT into the above cuvette + The absorption at 336 nm was detected using a UV-visible spectrophotometer.
[0119] 3) Inject 5 μL of recombinant enzyme (2 mg / ml) into the cuvette, measure the change in absorbance at 336 nm, and calculate its activity. The catalytic activity results of recombinases 1 to 4 and the comparative recombinant enzyme are shown in Table 1. The absorption change at 336 nm during substrate hydrogenation catalyzed by recombinase 1 is shown in the figure below. Figure 26 shown.
[0120] Table 1. Oxidation and hydrogenation activity tests of recombinant enzymes based on different monoferric hydrogenase models
[0121]
[0122]
[0123] The results in Table 1 show that the presence of the 5-methyl group of pyridine improves the oxidation and hydrogenation activities of the recombinant enzyme. The presence of the 3-methyl group of pyridine significantly improves the oxidation and hydrogenation activities of the recombinant enzyme, and the activity of recombinant enzyme 1 is approximately 30 times that of recombinant enzyme 3. Compared with the model recombinant conducted by the Seigo research group and Professor Hu Xile in 2015 (reference: S. Shima, D. Chen, T. Xu, MD Wodrich, T. Fujishiro, KM Schultz, J. Kahnt, K. Ataka and X. Hu, Nat. Chem. 2015, 7, 995-1002), the recombinant enzyme based on the monoferric hydrogenase model synthesized by the present invention has a huge improvement in catalytic activity, with the highest oxidation activity and hydrogenation activity reaching 19.5±1.3 U / mg and 21.1±2.5 U / mg, respectively, and has broad application prospects.
Claims
1. A type of monoferric hydrogenase model, characterized in that The chemical structure is shown below: where R 1 、R 2 are the same or different monodentate ligands or R 1 、R 2 represents a bidentate ligand, R 3 、R 5 The same or different, representing H or C1-C6 alkyl, R 4 represents H or hydroxyl.
2. The monoferrohydrogenase model according to claim 1, wherein The monodentate ligand is selected from NH3, F - 、Cl - Br - , I - 、S 2- 、SCN - 、NO 3- 、N3 - , acetonitrile, pyridine, triphenylphosphine, carbon monoxide, and the bidentate ligand is selected from ethylenediamine, oxalate, nitrite, 2-mercaptoethanol, 6-methyl-2-mercaptopyridine or acetate.
3. The monoferrohydrogenase model according to claim 1, wherein The R 1 、R 2 Same or different, selected from F - 、Cl - Br - or I - ; R 3 、R 5 The same or different, represents H, methyl or ethyl; R 4 represents H or hydroxyl.
4. The monoferrohydrogenase model according to claim 1, wherein The R 1 、R 2 Same, represents Cl - Br - or I - , R 3 、R 5 Same, represents H, methyl or ethyl, R 4 represents H or hydroxyl.
5. The monoferric hydrogenase model according to any one of claims 1 to 4, characterized in that The anion of the monoferrohydrogenase model substance forms a salt with the cation.
6. The monoferrohydrogenase model according to claim 5, characterized in that The cation is selected from NH4 + 、Na + , K + 、Ag + , tetrabutylammonium, tetraethylammonium.
7. A recombinant monoferric hydrogenase, characterized in that The monoferronihydrogenase model is recombined with the monoferronihydrogenase model according to any one of claims 1 to 6 and the natural monoferronihydrogenase with the active center removed.
8. The recombinant monoferrohydrogenase according to claim 7, characterized in that The monoferric hydrogenase is selected from one or more of M. jannaschii, Methanotorris igneus, Methanocaldococcus infernus, Methanococcusaeolicus, Methanolacinia paynteri, and Methanoregula formicica.
9. Use of the recombinant monoferric hydrogenase according to claim 7 or 8 in catalyzing hydrogen heterolytic cleavage reactions.
10. The use according to claim 9, characterized in that The recombinant monoferric hydrogenase catalyzes the oxidation reaction of methylene-H4MPT or catalyzes the oxidized methenyl-H4MPT + hydrogenation reaction.