KR102638205B1 - Bioconversion method for N-oxidizing isoquinoline alkaloids using CYP105D18 - Google Patents

Abstract

The present invention relates to a method for bioconversion of isoquinoline alkaloid compounds comprising the step of N-oxidizing isoquinoline alkaloid compounds using CYP105D18, and relates to a method for bioconversion of isoquinoline alkaloid compounds using the CYP105D18 enzyme according to the present invention. The oxidative bioconversion method has the advantage of being able to N-oxidize isoquinoline alkaloid compounds with high efficiency because CYP105D18 has high tolerance to hydrogen peroxide (H ₂ O ₂ ) and unique substrate specificity.

Description

Bioconversion method for N-oxidizing isoquinoline alkaloids using CYP105D18}

The present invention relates to a method for N-oxidation bioconversion of isoquinoline alkaloid compounds using CYP105D18 enzyme, and more specifically, to a method for bioconversion of isoquinoline alkaloid compounds comprising the step of N-oxidation of isoquinoline alkaloid compounds using CYP105D18. It's about method.

Cytochrome P450 (CYP) enzymes include heme b-containing enzymes expressed in a variety of species. These enzymes perform various catalytic reactions such as hydroxylation, alcohol and carbonyl oxidation, epoxidation, demethylation, N-oxidation, ring cleavage, coupling and formation of numerous endogenous and exogenous compounds such as steroids, fatty acids, xenobiotics and other drugs. Catalyzes the reaction.

CYPs are involved in regio- and stereospecific reactions along with substrate flexibility and are suitable targets for biotechnology applications. The complex mechanisms of CYP reactions involve specific redox partners and costly cofactors. Therefore, it essentially limits the broad utility of CYP.

However, some CYP monooxygenases and peroxygenases utilize oxygen surrogate systems such as hydrogen peroxide (H ₂ O 2 ) and organic surrogates such as cumene hydroperoxide or iodosylbenzene for efficient catalysis of various substrates. ) supports a shunt pathway using ).

As a prior art regarding a bioconversion method using enzymes, a method for bioconversion of ginsenosides through enzyme and lactic acid bacteria fermentation in red ginseng peel (Public Patent Publication No. 10-2021-0087536) is disclosed, but using enzymes There is no known method for bioconversion of steroids that includes the step of hydroxylating carbon 27 of the steroid.

Republic of Korea Patent Publication No. 10-2021-0087536

The present inventors conducted research to determine biochemical and structural information on CYP105 family proteins, found that isoquinoline alkaloid compounds can be N-oxidized using CYP105D18, and completed the present invention.

Therefore, the purpose of the present invention is to provide a method for N-oxidation bioconversion of isoquinoline alkaloid compounds using CYP105D18 enzyme.

In order to achieve the above object, the present invention provides a method for bioconversion of isoquinoline alkaloid compounds, including the step of N-oxidizing isoquinoline alkaloid compounds using CYP105D18.

In one embodiment of the present invention, the isoquinoline alkaloid compound may be any one selected from the group consisting of papaverine, codeine, and berberine.

In one embodiment of the present invention, the CYP105D18 may be cytochrome P450 derived from Streptomyces .

In one embodiment of the present invention, the CYP105D18 may be cytochrome P450 derived from Streptomyces laurentii .

In one embodiment of the present invention, the isoquinoline alkaloid compound is papaverine, the K _m value of CYP105D18 for papaverine may be 212 to 215 μM, and the K _cat value may be 4 to 6 min ^-1 .

In one embodiment of the present invention, CYP105D18 may be resistant to hydrogen peroxide (H ₂ O ₂ ).

In one embodiment of the present invention, the heme oxidation rate (k) of CYP105D18 may be as low as less than 0.3 min ^-1 even in the presence of a high concentration of 200mM hydrogen peroxide (H ₂ O ₂ ).

In one embodiment of the present invention, CYP105D18 has a bendable structure, and may have a high substrate preference for isoquinoline alkaloid compounds due to the structure.

Additionally, in order to achieve the above object, the present invention provides an isoquinoline alkaloid compound obtained by the above bioconversion method.

In one embodiment of the present invention, the isoquinoline alkaloid compound may be any one selected from the group consisting of papaverine, codeine, and berberine.

The N-oxidation bioconversion method of isoquinoline alkaloid compounds using the CYP105D18 enzyme according to the present invention produces isoquinoline alkaloid compounds with high efficiency because CYP105D18 has high tolerance to hydrogen peroxide (H ₂ O ₂ ) and unique substrate specificity. There is an advantage in that it can be N-oxidized.

In addition, the N-oxidation bioconversion method of isoquinoline alkaloid compounds using the CYP105D18 enzyme according to the present invention can be usefully used as a drug development method that can replace existing steroids in the pharmaceutical field.

Figures 1a and 1b show HPLC chromatograms and LC-MS spectra (inside box) for papaverine (Figure 1a) and tetrahydropalmatine (Figure 1b), where the reaction (top) was incubated in 40mM H ₂ for 1 hour. Performed with 400 μM of substrate and 3 μM of CYP105D18 in the presence of O ₂ , similar reaction conditions were used for controls (below), 3 μM of heat-inactivated CYP105D18 (positive control) and CYP105D18 (negative control) were used, positive and negative The control showed a similar HPLC chromatogram without the formation of any product.
Figure 2 shows CYP105D18 in the presence of H ₂ O ₂ at various concentrations ((a) 1mM, (b) 5mM, (c) 10mM, (d) 20mM, (e) 40mM, (f) 100mM and (g) 200mM). This shows the heme oxidation pattern, where the concentration of CYP was fixed at 3mM, the decrease in Soret peak (417 nm) was measured at 90-second intervals for 30 minutes, and papaverine N- in vitro by CYP105D18(h) Optimized reaction conditions for H ₂ O ₂ concentration for oxidation are 3 μM CYP, 400 μM substrate, and 5-800 mM H ₂ O ₂ ; Means and standard deviations are from three independent reactions under similar conditions.
Figure 3(a) shows the time-dependent conversion of papaverine to papaverine N-oxide. The conversion was measured every 5 minutes for 1 hour using 1 μM CYP, and the reaction was carried out in 40mM H ₂ O ₂ It starts by.
Figure 3 (b) shows a hyperbolic fit for papaverine N-oxide, and Figure 3 (c) shows a hyperbolic fit for tetrahydropalmatine (THP), with average K _m and k _cat values were calculated as the standard deviation of three independent experiments performed under similar conditions.
Figure 4(a) shows a ribbon diagram of the CYP105D18 structure, where rainbow colors from blue to red indicate the N-terminal to C-terminal positions of residues in the model, and the right panel is rotated 90 degrees with respect to the left panel to the right. Orient the panel's substrate binding funnel side toward the reader on the left panel. The region of the active site is indicated by orange sticks and conserved residues in the B/C loop and FG turn are indicated by red circles.
Figure 4 (b) shows another homologous CYP, CYP105D18 from S. laurentii , Streptomyces sp. CYP105D7 and CYP105D6/PteD from S. avermitilis , CYP105D6 from S. coelicolor , CYP105A1 from S. griseolus , CYP105AB3 from Nonomuraea recticatena , GfsF from S. graminofaciens , CYP105AS1 from Amycolatopsis orientalis and S. Multiple sequence alignment with CYP105P1 from avermitilis is shown, with secondary structural elements indicated and colored above the aligned sequence.
Figure 5 shows the superposition of the H ₂ O ₂ insensitive rabbit P450 4B1 and CYP105D18, where the residues corresponding to Cys448, Phe441 and Gln451 of P450 4B1 are indicated using sticks, and the heme and water molecules for CYP105D18 are shown. The 2Fo-Fc electron density map is shown as a gray mesh (outlined at the 2.0σ level), and the octane molecule of P450 4B1 is shown as a purple stick.
Figure 6 is a comparison of the structures of the substrate-free structure and the papaverine complex using CYP105D18. Figure 6 (a) shows substrate-free CYP105D18 and papaverine-bound CYP105D18 in blue and orange, respectively. , the turns of the BC loop and FG of CYP105D18 were shown in a ribbon diagram.
Figure 6 (b) shows an enlarged view of αB' and the hinge region of the active site, and (c) shows an enlarged view of the heme and papaverine binding site, with papaverine and interacting residues shown as stick models. The yellow stick model represents papaverine, and the bound heme molecule is also shown as a stick model.
Figure 7 shows the bending movement of papaverine at the substrate binding site of CYP105D18. Figure 7 (a) shows the detailed binding mode of CYP105D18 and papaverine, and papaverine is located at the bottom of the active site, and water molecules ( The sphere (green) exists between the dimethodiisoquinoline nitrogen and the iron of the heme group, and papaverine and heme are yellow and magenta, respectively.
Figure 7 (b) is an enlarged view of the binding pocket in (a), and the cross-sectional image of CYP105D18 was created using PyMol (Schrodinger).
Figure 8 is a comparison of bendable and non-bendable substrates, where papaverine and heme molecules co-crystallized with CYP105D18 are shown as yellow and orange sticks, and after docking calculations, each potential substrate is represented by a different color code. Indicated by sticks, the distance between the heme of CYP105D18 and the water molecule interacting with the nitrogen is indicated in each figure, the docking experiments were performed with the Lamarckian genetic algorithm (LGA) method in Autodock Vina (Trott & Olson, 2010). did.

In the present invention, “bioconversion method” refers to a method that can produce a new product through biotechnology technologies such as microbial fermentation and enzyme treatment or replace existing chemicals synthesized and produced by existing chemical synthesis processes. It refers to technology and is also called bioconversion, biotransformation, biosynthesis, bio-catalysis, etc.

A first embodiment of the present invention relates to a method for bioconversion of an isoquinoline alkaloid compound comprising the step of N-oxidizing the isoquinoline alkaloid compound using CYP105D18.

The isoquinoline alkaloid compound may be any one selected from the group consisting of papaverine, codeine, and berberine, but is not limited thereto.

The papaverine is an isoquinoline alkaloid contained in opium, and because it causes smooth muscle relaxation, it has been used to treat vasospasm. Papaveine has also been administered as an alternative to NSAIDs in the treatment of renal colic, migraine, schizophrenia, cancer, and even viral infections in some patients. Despite its medical importance, this compound of papaverine has many side effects. .

However, papaverine modified by the bioconversion method of the present invention has the advantages of better efficacy, reduced cytotoxicity, and can be easily metabolized by the body.

The codeine is a drug that relieves pain and suppresses coughing. It reduces pain by blocking the transmission of pain from the periphery to the central region, and reduces the occurrence of coughing by suppressing the coughing center in the brain. It is used for addiction, misuse, or Because there is a risk of abuse, it must be used under the regular management and supervision of a medical professional and is designated as a narcotic.

The berberine is a natural plant alkaloid contained in the bark, roots, and stems of barberry and yellow lotus, and is known as a natural antibiotic and antioxidant.

The CYP105D18 may be a cytochrome P450 derived from Streptomyces , preferably from Streptomyces laurentii , but is not limited thereto.

The isoquinoline alkaloid compound may be papaverine, and the K _m value of CYP105D18 for papaverine may be 212-215 μM, preferably 213-214 μM, more preferably 213 ± 26 μM, K _{The cat} value may be 4 to 6 min ^-1 , preferably 4.5 to 5.5 min ^-1 , and more preferably 5 ± 0.3 min ^-1 , but is not limited thereto.

The CYP105D18 may be resistant to hydrogen peroxide (H ₂ O ₂ ), and more specifically, the heme oxidation rate (k) of the CYP105D18 is 0.3 min even in the presence of a high concentration of 200mM hydrogen peroxide (H ₂ O ₂ ) ^. It may be as low as less than ¹ , but is not limited to this.

The CYP105D18 has a bendable structure, and due to the above structure, it may have a high substrate preference for isoquinoline alkaloid compounds.

In addition, the present invention provides, as a second embodiment, an isoquinoline alkaloid compound obtained by the above bioconversion method.

The isoquinoline alkaloid compound may be any one selected from the group consisting of papaverine, codeine, and berberine, but is not limited thereto.

Since papaverine, codeine, and berberine are the same as described in the first embodiment, detailed description thereof will be omitted.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example 1> Functional characterization of CYP105D18

Although in vitro biotransformation was not confirmed through the interaction of CYP105D18 with Pdx/PdR or Fdx/RdR, in vitro biotransformation using H ₂ O ₂ , an oxygen counterpart, supported N-oxidation of papaverine and hydroxylation of THP ( Figure 1a).

We report the first CYP105 subfamily enzyme showing tolerance to H ₂ O ₂ , which was analyzed by HPLC and LC-MS, and Papaverine N-oxide was characterized using nuclear magnetic resonance analysis. However, THP did not conduct structural analysis because the conversion rate under optimal conditions was less than 12%.

Substrate binding experiments were performed to support the in vitro and in vivo transformation of papaverine by CYP105D18. Among the four substrates, only papaverine induced a spectral shift at 422 nm (Figure 1b). This is the first report on compound N-oxidation by bacterial CYP, and compared to existing experiments, CYP105D18 efficiently converts papaverine to papaverine N-oxide in the presence of a stoichiometric amount of H ₂ O ₂ , demonstrating the use of chemical methods. It has been shown to provide important insights.

<Example 2> H of CYP105D18 ₂ O ₂ Stability and Optimization

Only H ₂ O ₂ supported CYP105D18-dependent N-oxidation of papaverine. Therefore, H ₂ O ₂ Oxidative degradation of heme was analyzed depending on concentration. The rate constant k was low even in the presence of high concentrations of H ₂ O ₂ , suggesting a high tolerance capacity of CYP105D18 to H ₂ O ₂ (Figure 2).

The time-dependent in vitro conversion of papaverine N-oxide by CYP105D18 is shown in Figure 3(a). The K _m and K _cat values for papaverine are estimated to be 213±26μM and 5±0.3 min ^-1 , respectively, and for tetrahydropalmatine (THP), they are 75±15μM and 0.13±0.01 min ^-1 , respectively. It was confirmed that the catalyst efficiency was higher.

The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 sec ^-1 μM ^-1 , which was much higher than the catalytic efficiency of THP, which was 0.11 sec ^-1 μM ^-1 (Figure 3).

The heme oxidation rate (k) of CYP105D18 was low (<0.3 min ^-1 ) even in the presence of high concentration (200mM) of H ₂ O ₂ . The high _H2O2 tolerance capacity _of CYP105D18 resulted in a higher turnover rate prior to heme oxidation.

<Example 3> Substrate-free structure of CYP105D18

To obtain detailed structural information related to the active site, a structural investigation of CYP105D18 was performed. The final model obtained electron densities for most of the molecule except for the N-terminus, the loop containing the residues connecting αB and αC, and the asymmetric unit has the CYP105D18 molecule, which also appears as a monomer in size exclusion chromatography analysis. It was shown that it exists (Figure 4(a)). It was deposited in the Protein Data Bank as PDB entry 7DI3.

As can be observed in the structure of the CYP105 family, it can be divided into two units, the β-sheet and the αC-αJ helix, and forms a deep, concave funnel-shaped active site located in the center between the β-sheet and the helix-rich unit. do. This active site consists of a B/C loop (residues 173-187), a turn region between αF and αG, and a C-terminal loop (residues 336-396).

Although it shares high structural homology with the CYP105 family, residues in the three regions are hardly conserved (Figure 4(b)). This suggests that this part is essential for substrate selectivity for the CYP family, and in previous studies of CYP105D7, arginine residues (Arg70, Arg88, Arg190) in the B/C loop and αG in the substrate binding pocket are conserved in the CYP105 family, indicating substrate recognition. It was shown that it can be an important residue in .

CYP105D18 contains Ser63 and Phe184, which correspond to Arg70 and Arg190 of CYP105D7, suggesting that substrate residue recognition in the CYP active site is more complex and diverse.

<Example 4> H of CYP105D18 ₂ O ₂ Structural features for stability

Molecular dynamics simulations based on rabbit P450 4B1 suggested that Gln451 of P450 4B1 is an essential residue for P450 thiol sensitivity. It showed heme thiolate and sulfenic acid dependent open and closed structures.

In addition, CYP105D18 shows high sequence and structural similarity to P450 4B1, has Phe338 and Gln348 corresponding to Phe441 and Gln451 of P450 4B1, and is in a closed form. This suggests that a similar mechanism to the P450 4B1 access region to the heme region of CYP105D18 may involve structural changes upon oxidation, and that this closed structure results in the H ₂ O ₂ resistance of CYP105D18 (Figure 5).

<Example 5> Comparison of substrate binding pockets between the substrate-free structure of CYP105D18 and the papaverine complex structure

In addition to the substrate-free structure of CYP105D18, the papaverine complex structure (PDB entry 7LDS) was determined to support papaverine N-oxidation and to understand the substrate binding mode and structural changes that occur upon binding. Structural comparison of the conformations with and without substrates showed that structural changes mainly occurred in the B/C loop region.

A flexible and less structured loop was observed in the B/C loop, and the electron density for residues 73-84 could not be observed in a substrate-free structure, indicating that this region has high flexibility.

Overall, this shows that the B/C loop is flexible and remains open and identified without substrate, and this region is believed to be responsible for substrate binding and specificity (Figure 6(a)).

Papaverine was observed to be buried in the internal hydrophobic cavity of CYP105D18, surrounded by residues Ala85, Leu87, Val231, Ala282, Val234 and Ile386, and devoid of hydrophilic residues.

The dimethoxyisoquinoline and dimethoxy-phenyl groups are bent about 90 degrees by a single carbon bond, which is linked using two single carbon bonds that other isoquinoline alkaloids cannot bend. Therefore, although the overall biochemical properties of the tested substrates were similar, the biochemical data strongly support that CYP105D18 selectively modified papaverine (Figure 6(b)).

The distance between water and nitrogen was 3.2 A˚, and the distance between water and iron was 2.1 A˚, indicating that the binding mode represents the true mode of N-oxidation (Figure 7). To further support the above observations, molecular docking simulations were performed using bendable and non-bendable substrates.

The reliability of the docking simulation was confirmed using the pacaverine model, and the simulation results showed that the nitrogen in the bendable chemical was closer to the water molecule interacting with the iron of the heme molecule in CYP105D18 compared to the non-bendable compound. (Figure 8), and it is believed that the bending motion of the ligand may be an important function in setting the distance and coordinates while the hydroxyl group moves.

As the specific parts of the present invention have been described in detail above, those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (10)

A method for bioconversion of an isoquinoline alkaloid compound comprising the step of N-oxidizing the isoquinoline alkaloid compound using CYP105D18.
The method of claim 1, wherein the isoquinoline alkaloid compound is any one selected from the group consisting of papaverine, codeine, and berberine.
The method for bioconversion of an isoquinoline alkaloid compound according to claim 1, wherein CYP105D18 is cytochrome P450 from Streptomyces .
The method for bioconversion of an isoquinoline alkaloid compound according to claim 1, wherein CYP105D18 is cytochrome P450 derived from Streptomyces laurentii .
The isoquinoline alkaloid compound of claim 1, wherein the isoquinoline alkaloid compound is papaverine, and the K _m value of CYP105D18 for papaverine is 212-215 μM, and the K _cat value is 4-6 min ^-1 . Method for bioconversion of quinoline alkaloid compounds.
The method for bioconversion of an isoquinoline alkaloid compound according to claim 1, wherein CYP105D18 is resistant to hydrogen peroxide (H ₂ O ₂ ).
The method of claim 6, wherein the heme oxidation rate (k) of CYP105D18 is as low as less than 0.3 min ^-1 even in the presence of a high concentration of 200mM hydrogen peroxide (H ₂ O ₂ ). .
The method for bioconversion of isoquinoline alkaloid compounds according to claim 1, wherein CYP105D18 has a bendable structure and has a high substrate preference for isoquinoline alkaloid compounds due to the structure.
An isoquinoline alkaloid compound obtained by the bioconversion method of any one of claims 1 to 8.
The isoquinoline alkaloid compound according to claim 9, wherein the isoquinoline alkaloid compound is any one selected from the group consisting of papaverine, codeine, and berberine.