An enzyme composition for synthesizing creatmycin derivatives and its application

Abstract

The present invention provides an enzyme composition for synthesizing creatmycin derivatives and its application. The enzyme composition contains Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases. The enzyme composition provided by the present invention uses tryptophan derivatives as substrates to synthesize norcreatmycin derivatives through the multi-enzyme combination system of the present invention. The present invention solves the method of synthesizing norcreatmycin derivatives using other tryptophan derivatives. Compared with chemical synthesis, enzymatic synthesis has the advantages of strong specificity, mild and easy-to-control reaction conditions, simple operation, and environmental friendliness.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthesis of innovative mycin derivatives, and particularly relates to an enzyme composition for synthesizing innovative mycin derivatives and applications thereof. Background Art

[0002] Innovative mycin is a novel antibiotic discovered by Chinese scientists in the 1970s from Actinomycetes spp. in Jinan. It possesses a unique indole-dihydrothiopyran ring nucleus structure and a novel sulfur atom introduction mechanism. As a tryptophan analog, innovative mycin can target and inhibit bacterial tryptophanyl-tRNA synthetase. Experimental studies have shown significant inhibitory effects against Escherichia coli and Shigella spp., but no inhibitory effect against ovine tryptophanyl-tRNA synthetase, making it a candidate molecule for a new antibiotic. However, its narrow antimicrobial spectrum has limited its widespread clinical use.

[0003] Currently, the main method for modifying innovamycin to obtain innovamycin derivatives is chemical synthesis. However, chemical synthesis methods are cumbersome, the reaction conditions are complex and difficult to control, and some environmentally polluting reagents such as ethyl mercaptoacetate are inevitably required during chemical synthesis. Summary of the Invention

[0004] The purpose of the present invention is to provide an enzyme composition for synthesizing innovamycin derivatives and its application, as well as a method for synthesizing innovamycin derivatives using a tryptophan derivative as a substrate through the enzyme composition of the present invention, thereby providing a lead compound resource for screening innovamycin derivatives with good antibacterial activity.

[0005] The present invention first provides an enzyme composition, which comprises Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases;

[0006] The amino acid sequence of the Cxm3 protease is SEQ ID NO: 1, the amino acid sequence of the Cxm4 protease is SEQ ID NO: 2, the amino acid sequence of the Cxm5 protease is SEQ ID NO: 3, the amino acid sequence of the Cxm6 protease is SEQ ID NO: 4, the amino acid sequence of the Cxm7 protease is SEQ ID NO: 5, the amino acid sequence of the CxmM protease is SEQ ID NO: 6, the amino acid sequence of the FdR protease is SEQ ID NO: 7, and the amino acid sequence of the Fdx protease is SEQ ID NO: 8. However, homologous enzymes / isozymes with the proteins with sequences of SEQ ID NOs: 1-8 can also be used for catalysis.

[0007] The enzyme composition provided by the present invention is used to synthesize norcreatinomycin derivatives with tryptophan derivatives as substrates through the multi-enzyme combination system of the present invention;

[0008] The tryptophan derivatives are 5-fluorotryptophan (1), 5-chlorotryptophan (2), 5-bromotryptophan (3), 5-methyltryptophan (4), 6-fluorotryptophan (5), 6-chlorotryptophan (6), 6-bromotryptophan (7), 6-methyltryptophan (8), and 7-methoxytryptophan (9).

[0009] The norcreatinomycin derivatives are 5-fluoronorcreatinomycin (a), 5-chloronorcreatinomycin (b), 5-bromonorcreatinomycin (c), 5-methylnorcreatinomycin (d), 6-fluoronorcreatinomycin (e), 6-chloronorcreatinomycin (f), 6-bromonorcreatinomycin (g), 6-methylnorcreatinomycin (h), and 7-methoxynorcreatinomycin (i).

[0010] As described in the examples, norcreatinomycin derivatives a-i are prepared from tryptophan derivatives 1-9.

[0011] The method of the present invention is to synthesize creatinomycin derivatives by adding tryptophan derivatives, the above-mentioned enzyme composition, NADPH, ATP, Na2S2O3, MgCl2, sodium pyruvate, pyridoxal phosphate (PLP), and DTT to the reaction buffer;

[0012] One composition of the reaction buffer is as follows: 50 mM NaH2PO4, 300 mM NaCl, pH 8.0.

[0013] The present invention solves the method of synthesizing norcreatinomycin derivatives using other tryptophan derivatives. Compared with chemical synthesis, enzymatic synthesis has the advantages of strong specificity, mild and easy-to-control reaction conditions, simple operation, and environmental friendliness. Description of the Drawings

[0014] Figure 1 : Chemical structure diagram of tryptophan derivatives,

[0015] Figure 2 : Structure and HPLC-MS detection spectrum of 5-fluoronorcreatinomycin,

[0016] Figure 3 : Structure and HPLC-MS detection spectrum of 5-chloronorcreatinomycin,

[0017] Figure 4 : Structure and HPLC-MS detection spectrum of 5-bromonorcreatinomycin,

[0018] Figure 5: Structure of 5-methyl demethylcreatmycin and HPLC-MS detection spectrum,

[0019] Figure 6 : Structure of 6-fluoro demethylcreatmycin and HPLC-MS detection spectrum,

[0020] Figure 7 : Structure of 6-chloro demethylcreatmycin and HPLC-MS detection spectrum,

[0021] Figure 8 : Structure of 6-bromo demethylcreatmycin and HPLC-MS detection spectrum,

[0022] Figure 9 : Structure of 6-methyl demethylcreatmycin and HPLC-MS detection spectrum,

[0023] Figure 10 : Structure of 7-methoxy demethylcreatmycin and HPLC-MS detection spectrum. Detailed implementation mode

[0024] The present invention first provides an enzyme composition, and the enzyme composition contains Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases;

[0025] Among them, the amino acid sequence of Cxm3 is SEQ ID NO:1, the amino acid sequence of Cxm4 is SEQ ID NO:2, the amino acid sequence of Cxm5 is SEQ ID NO:3, the amino acid sequence of Cxm6 is SEQ ID NO:4, the amino acid sequence of Cxm7 is SEQ ID NO:5, the amino acid sequence of CxmM is SEQ ID NO:6, the amino acid sequence of FdR is SEQ ID NO:7, and the amino acid sequence of Fdx is SEQ ID NO:8.

[0026] However, homologous enzymes / isoenzymes of the proteins with sequences of SEQ ID NO:1-8 can also be used for catalysis.

[0027] The enzyme composition provided by the present invention can realize the synthesis of demethylcreatmycin derivatives through an in vitro "one-pot method" with tryptophan derivatives as substrates.

[0028] Among them, the tryptophan derivatives (the structure is shown in Figure 1 ) are mainly halogen atoms (F, Cl, Br) and methyl substitution at the C5 and C6 positions of the indole ring and C7 oxygen-methyl substitution.

[0029] The synthesis method described above involves adding tryptophan derivatives, the enzyme composition described above, NADPH, ATP, Na2S2O3, MgCl2, sodium pyruvate, pyridoxal phosphate (PLP), and DTT to a reaction buffer to synthesize various norcreatinine derivatives.

[0030] The reaction buffer described above is a salt solution that can ensure the enzymatic reaction activity and the solubility of other added substances. One specific component is 50 mM NaH2PO4, 100 - 300 mM NaCl, 0 - 10% glycerol, pH 7.0 - 8.0. However, other buffers that can exert the efficacy of the enzyme composition can also be used.

[0031] The concentrations of the enzyme composition and other cofactor components are 1 - 10 μM Cxm3, 1 - 10 μM Cxm4, 1 - 10 μM Cxm5, 1 - 10 μM Cxm6, 1 - 10 μM Cxm7, 1 - 10 μM CxmM, 5 - 50 μM FdR, 10 - 100 μM Fdx, 1 - 10 mM NADPH, 0.5 - 2 mM ATP, 0.5 - 2 mM Na2S2O3, 1 - 5 mM MgCl2, 1 - 3 mM sodium pyruvate, 1 - 20 μM pyridoxal phosphate (PLP), and 1 - 5 mM DTT.

[0032] The present invention will be described in detail below with reference to examples and the accompanying drawings.

[0033] Example 1: "One-pot" synthesis of 5-fluoronorcreatinine

[0034] (1) Induction and expression of proteins

[0035] The Escherichia coli strains highly expressing Cxm3, Cxm4, Cxm5, Cxm6, CxmM, Fdx, and FdR proteins were streaked on an LB plate (containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol), and the Escherichia coli highly expressing Cxm7 protein was inoculated on an LB plate containing 50 μg / mL kanamycin, and cultured at 37°C for 24 h. Single colonies were picked and inoculated into 50 mL of LB medium containing 50 μg / mL kanamycin or 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and cultured overnight at 37°C and 220 rpm on a shaker. Inoculated into 500 mL of TB or LB medium with the corresponding resistance at an inoculation amount of 1%, and cultured at 37°C and 200 rpm until the OD600 was between 0.8 and 1. Then, IPTG with a final concentration of 200 μM, 5-aminolevulinic acid (5-ALA) with a concentration of 500 μM, and vitamin B1 (VB1) with a concentration of 500 μM were added. Subsequently, the protein expression was induced by culturing at 16°C and 150 rpm for 18 - 20 h.

[0036] (2) Purification of proteins

[0037] Centrifuge to collect the bacteria cells, resuspend the cells by vortexing with Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), ultrasonically disrupt the cells, then centrifuge at 10000 rpm for 60 min at 4 °C. Incubate the supernatant with Ni-NTA at 4 °C for 60 min. Then, elute the impurity proteins using Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0); elute the target protein using Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), select an appropriate ultrafiltration tube to concentrate the protein solution according to the size of the target protein; finally, use a PD-10 desalting column to remove imidazole, and use Desalting buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0) as the buffer solution.

[0038] (3) Synthesis of 5-fluoro-demethyl-innovamycin by "one-pot" reaction

[0039] Using 1 mM 5-fluorotryptophan (1) as the substrate, add 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP) and 5 mM DTT. The reaction buffer is 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, add 2 volumes of methanol to the reaction solution to terminate the reaction.

[0040] The reaction product was detected by HPLC-MS. The results showed that after multi-enzyme catalysis of compound 1, 5-fluoro-demethyl-innovamycin with the expected molecular weight was correspondingly produced, that is, the absorption peak a and the quasi-molecular ion peak m / z 238.0341 [M+H] + ( Figure 2 ), which is consistent with the molecular weight of 238.0338 [M+H] of the fluoro-demethyl-innovamycin derivative.

[0041] Example 2: "One-pot" synthesis of 5-chloro-demethyl-innovamycin

[0042] Using 1 mM 5-chlorotryptophan (2) as the substrate, add 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP) and 5 mM DTT. The reaction buffer is 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, add 2 volumes of methanol to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, showing that after multi-enzyme catalysis of compound 2, 5-chloronorcreatinine corresponding to the expected molecular weight was generated, that is, the quasi-molecular ion peak of absorption peak b was m / z 254.0047 [M+H] + ( Figure 3 ), which is consistent with the molecular weight of the chloronorcreatinine derivative 254.0043 [M+H] + Consistent

[0043] Example 3: "One-pot" synthesis of 5-bromonorcreatinine

[0044] Using 1 mM 5-bromotryptophan (3) as the substrate, add 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP) and 5 mM DTT. The reaction buffer is 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, add 2 volumes of methanol to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, showing that after multi-enzyme catalysis of compound 3, 5-bromonorcreatinine corresponding to the expected molecular weight was generated, that is, the quasi-molecular ion peak of absorption peak c was m / z 297.9537 [M+H] + ( Figure 4 ), which is consistent with the molecular weight of the bromonorcreatinine derivative 297.9537 [M+H] + Consistent

[0045] Example 4: "One-pot" synthesis of 5-methylnorcreatinine

[0046] Using 1 mM 5-methyltryptophan (4) as the substrate, add 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP), and 5 mM DTT. The reaction buffer is 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, add 2 volumes of methanol to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, showing that after multi-enzyme catalysis of compound 4, 5-methyl demethyl-innovamycin corresponding to the expected molecular weight was produced, that is, the absorption peak d and the quasi-molecular ion peak m / z 234.0592 [M+H] + ( Figure 5 ), which is consistent with the molecular weight of the methylated demethyl-innovamycin derivative 234.0589 [M+H] + Consistent.

[0047] Example 5: "One-pot" synthesis of 6-fluorodemethyl-innovamycin

[0048] Using 1 mM 6-fluorotryptophan (5) as the substrate, add 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP), and 5 mM DTT. The reaction buffer is 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, add 2 volumes of methanol to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, and the results showed that after multi-enzyme catalysis of compound 5, 5-fluorodemethyl-innovamycin corresponding to the expected molecular weight was produced, that is, the absorption peak e and the quasi-molecular ion peak m / z 238.0333 [M+H] + ( Figure 6 ), which is consistent with the molecular weight of the fluorinated demethyl-innovamycin derivative 238.0338 [M+H] + Consistent.

[0049] Example 6: "One-pot" synthesis of 6-chlorodemethyl-innovamycin

[0050] 1mM 6-chlorotryptophan (6) was used as the substrate, and 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2S2O3, 5mMMgCl2, 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT were added. The reaction buffer was NaH2PO4 50mM, NaCl 300mM, pH 8.0, with a total volume of 100μL. After reacting at 30℃ for 6h, 2 volumes of methanol were added to the reaction solution to terminate the reaction. The reaction products were detected by HPLC-MS. The results showed that compound 6 produced 6-chlorodemethylinnovamycin with the expected molecular weight after multi-enzyme catalysis, namely, the absorption peak f quasi-molecular ion peak m / z 254.0040 [M+H] + ( Figure 7 ), and the molecular weight of the chlorodemethylinnovative mycin derivative is 254.0043 [M+H] + consistent.

[0051] Example 7: One-pot synthesis of 6-bromonortelocycline

[0052] 1mM 6-bromotryptophan (7) was used as the substrate, and 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2S2O3, 5mMMgCl2, 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT were added. The reaction buffer was 50mM NaH2PO4, 300mM NaCl, pH 8.0, and the total volume was 100μL. After reacting at 30℃ for 6h, 2 volumes of methanol were added to the reaction solution to terminate the reaction. The reaction products were detected by HPLC-MS, which showed that compound 7 produced 6-bromodemethylinnovamycin with the expected molecular weight after multi-enzyme catalysis, namely the absorption peak g quasi-molecular ion peak m / z 297.9540 [M+H] + ( Figure 8 ), and the molecular weight of the bromo-demethyl-innovative mycin derivative is 297.9537 [M+H] + Consistent.

[0053] Example 8: One-pot synthesis of 6-methylnorinnovamycin

[0054] Using 1 mM 6-methyltryptophan (8) as the substrate, 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP), and 5 mM DTT were added. The reaction buffer was 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, twice the volume of methanol was added to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, showing that compound 8 was catalytically converted by multiple enzymes to produce 6-methylnordeoxynivalenol with a molecular weight consistent with the expected value, i.e., absorption peak h, the quasi-molecular ion peak m / z 234.0592 [M+H]. + ( Figure 9 ), which is consistent with the molecular weight of the methylated nordeoxynivalenol derivative, 234.0589 [M+H]. + Consistent.

[0055] Example 9: "One-pot" synthesis of 7-methoxynordeoxynivalenol

[0056] Using 1 mM 7-methoxytryptophan (9) as the substrate, 10 μM Cxm3, 10 μM Cxm4, 10 μM Cxm5, 10 μM Cxm6, 10 μM Cxm7, 10 μM CxmM, 50 μM FdR, 100 μM Fdx, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2, 3 mM sodium pyruvate, 20 μM pyridoxal phosphate (PLP), and 5 mM DTT were added. The reaction buffer was 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, with a total volume of 100 μL. After reacting at 30 °C for 6 h, twice the volume of methanol was added to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, showing that compound 9 was catalytically converted by multiple enzymes to produce 6-methylnordeoxynivalenol with a molecular weight consistent with the expected value, i.e., absorption peak i, the quasi-molecular ion peak m / z 250.0537 [M+H]. + ( Figure 10 ), which is consistent with the molecular weight of the methylated nordeoxynivalenol derivative, 250.0538 [M+H]. + Consistent.

Claims

1. An enzyme composition, characterized in that, The enzyme composition described above comprises Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases; the amino acid sequence of the Cxm3 protease is SEQ ID NO:1, the amino acid sequence of the Cxm4 protease is SEQ ID NO:2, the amino acid sequence of the Cxm5 protease is SEQ ID NO:3, the amino acid sequence of the Cxm6 protease is SEQ ID NO:4, the amino acid sequence of the Cxm7 protease is SEQ ID NO:5, the amino acid sequence of the CxmM protease is SEQ ID NO:6, the amino acid sequence of the FdR protease is SEQ ID NO:7, and the amino acid sequence of the Fdx protease is SEQ ID NO:

8.

2. Use of the enzyme composition according to claim 1 in the synthesis of norcreatinomycin derivatives using tryptophan derivatives as substrates; the tryptophan derivatives are 5-fluorotryptophan, 5-chlorotryptophan, 5-bromotryptophan, 5-methyltryptophan, 6-fluorotryptophan, 6-chlorotryptophan, 6-bromotryptophan, 6-methyltryptophan or 7-methoxytryptophan; The norcreatinomycin derivatives are 5-fluoronorcreatinomycin, 5-chloronorcreatinomycin, 5-bromonorcreatinomycin, 5-methylnorcreatinomycin, 6-fluoronorcreatinomycin, 6-chloronorcreatinomycin, 6-bromonorcreatinomycin, 6-methylnorcreatinomycin or 7-methoxynorcreatinomycin.

3. A method for preparing norcreatinine derivatives from tryptophan derivatives, characterized in that, The method described above is to use the enzyme composition according to claim 1 for catalysis; The tryptophan derivatives are 5-fluorotryptophan, 5-chlorotryptophan, 5-bromotryptophan, 5-methyltryptophan, 6-fluorotryptophan, 6-chlorotryptophan, 6-bromotryptophan, 6-methyltryptophan or 7-methoxytryptophan; The norcreatinomycin derivatives are 5-fluoronorcreatinomycin, 5-chloronorcreatinomycin, 5-bromonorcreatinomycin, 5-methylnorcreatinomycin, 6-fluoronorcreatinomycin, 6-chloronorcreatinomycin, 6-bromonorcreatinomycin, 6-methylnorcreatinomycin or 7-methoxynorcreatinomycin; The method described above is to add tryptophan derivatives, enzyme composition, NADPH, ATP, Na2S2O3, MgCl2, sodium pyruvate, pyridoxal phosphate and DTT to the reaction buffer for the synthesis of creatinomycin derivatives.

4. The method according to claim 3, wherein The reaction buffer has the following composition: 50 mM NaH2PO4, 300 mM NaCl, pH 8.0.

Images