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P450 oxidase has the ability to selectively activate hydrocarbon bonds and oxidize them to hydroxyl and carboxyl groups. Generally, P450 oxidase only has good catalytic activity and high regional and stereoselectivity for natural substrates. These enzymes play an important role in the biosynthesis of natural products. However, plant P450 enzyme is not easy to express and purify after recombination, so it faces great challenges in application. Recently, Li Chun from Beijing University of science and technology and Tsinghua University, Yu Yang from Beijing University of science and technology and Wang Ying from Beijing University of science and technology modified cyp72a6, P450 oxidase, through experiments and calculation simulation, respectively, to obtain alcohol, aldehyde and carboxylic acid as the main products, as well as one of the isomers. Relevant research results were published on ACS catalyst (DOI: 10.1021 / acscalal. 0c00128).
Firstly, the author expressed the gene of 11 oxo - β - amyrin and cyp72a6 in synv to obtain the products with different oxidation degree at c-30 and the carboxylic acid oxidized at C-29 (Figure 1a). Using compound 11 oxo - β - amyrin as the substrate, the authors found that the c-30 position of wild-type cyp72a6 was closer to the ferrous center than the C-29 position (Figure 1b), and the distance was in the catalytic range, which was consistent with the experimental results.
Figure 1. (A) GC?MS spectrum showing the product distribution harboring wild type CYP72A63; (B) Structure model showing 11-oxo-β-amyrin in wild type CYP72A63; (C) Structure model showing glycyrrhetol in the wild type CYP72A6 .
(source: ACS catalyst.)
However, when using alcohol as the substrate, the author found that the hydrophobic interaction between the c-30 region and t338 made the c-30 region far away from the ferrous Center (Figure 1c). Based on the simulation results, the amino acids (l330, l333, t338 and A334) which may affect the hydrophobic effect in the active pocket were selected for mutation, and cyp72a63 (t338s) was finally screened out. The product of c-30 oxidation to acid and 5.8% aldehyde (Figure 2C) were obtained. In the same way, the selectivity of cyp72a63 (t338s) was studied by means of computational simulation. It was found that 11 oxo - β - amyrin had some deflections in the model, which made the distance difference between c-30 and C-29 with ferrous center larger, and the angle between two carbon atoms and ferrous center smaller (Figure 2A). By using alcohol as the substrate, it can be found that the c-30 position is at a suitable angle and distance, so that the substrate can be further oxidized to acid.
Figure 2. (A) Structure model of CYP72A63(T338S) with 11-oxo-β- amyrin; (B) Structure model of CYP72A63 (T338S) with glycyrrhetol; (C) GC?MS spectrum showing the product distribution in yeast strain harboring CYP72A63 (T338S).
(source: ACS catalyst.)
Then, the author began to study how to keep the main product in alcohol. The author thought that amino acids far away from the active pocket might participate in the recognition and binding of the substrate, and mutation of them might affect the binding between alcohol and enzyme, thus affecting the further oxidation process. Similarly, based on the computational simulation, the authors selected l509, l137 and l242 for mutation. Although l509h / l509n obtained a larger proportion of alcohol, the final titer was very small, which may be because the hydrophilic side chain interfered with the substrate binding. Finally, l509i got the best result (Figure 3B, table 1).
Figure 3. (A) L509 locates in the distal active pocket; (B) GC?MS spectrum showing the product distribution in yeast strain harboring CYP72A63 (L509I).
(source: ACS catalyst.)
After that, the author thinks that we can regulate different P450 reductase to affect the process of electron transfer and substrate binding, so as to obtain aldehyde as the main product. Through experimental screening, the author found that the combination of cyp72a63 (t338s) and gucpr2 got the best results, and the titer was 31.4 ± 5.8 mg / L. （Figure 4， Table 2）。 At the same time, this is the first example of P450 oxidation to produce aldehydes with high selectivity.
Figure 4. GC?MS spectrum showing the product distribution in yeast strain harboring CYP72A63 (T338S) and GuCPR2.
(source: ACS catalyst.)
Based on the above results, the author believes that if the substrate and enzyme active pocket are combined in such a way that the C-29 can be oriented to the ferrous center, it is possible to obtain the oxidation product at the C-29 position. It is predicted that L398 and l149 located on both sides of E-ring may play a role in the substrate rotation to some extent. In order to verify this assumption, the authors carried out site saturation mutations at these two positions and found that l398i can produce 100% C-29 oxidized carboxylic acid (Figure 5C). In order to understand the mechanism of high regioselectivity, the authors calculated and simulated the mutation, and found that the substrate rotated nearly 180 degrees, making C-29 more close to the ferrous Center (Figure 5b).
Figure 5. (A) Substrate pose in wild type CYP72A63; (B) Substrate pose in mutant CYP72A63(L398I); (C) GC?MS spectrum showing the product distribution in yeast strain harboring CYP72A63 (L398I).
(source: ACS catalyst.)
Finally, the efficiency of the catalyst was further improved by adjusting the proton transfer process. First of all, the author added molecular oxygen to the model through calculation and simulation, and found that the hydroxy in 338 threonine of wild-type cyp72a63 points away from molecular oxygen (Figure 6a), while the hydroxy in serine of mutant t338s points to the active center (Figure 6b). This difference makes the efficiency of proton transfer between alcohol and oxygen different, thus affecting the catalytic activity. The authors speculate that k236, e337 and s338 may be involved in this process (Figure 6c). On the other hand, when these amino acids are mutated into alanine, the catalytic activity is lost, which also confirms that these amino acids are necessary in the proton transfer process. By studying the amino acids around these amino acids, the author found that w205 may hinder the conformation change of k236, thus affecting the catalytic activity, so it was mutated. When it was mutated to alanine, because the restriction on k236 was relieved, the final cyp72a63 (t338s / w205a) increased the yield by 21.3%, and the titer reached 36.4 ± 3.0 mg / L. Based on the same strategy, the yields of aldehydes and alcohols increased by 59.6% and 8.2% respectively (Figure 6D).
Figure 6. (A) Binding pattern of 11-oxo-β-amyrin in wild type CYP72A63 with heme bonded oxygen molecule; (B) Binding pattern of 11-oxo-β-amyrin in CYP72A63 (T338S) with heme- bonded oxygen molecule; (C) Proton transfer pathway; (D) Increase rate of rare triterpenoids through strengthening proton delivery by mutant W205A.
(source: ACS catalyst.)
In a word, the author cleverly controlled the oxidation degree and regioselectivity of the substrate by computational simulation combined with directed evolution, including: (1) by adjusting the affinity between enzyme cavity and substrate, and the orientation of substrate in the cavity, the main product was carboxylic acid; (2) by weakening the affinity between alcohol and substrate, it could not be oxidized; (3) by adjusting the appropriate P450 The main product of protoenzyme is aldehyde; (4) the key residue is mutated to rotate the substrate to obtain isomer; (5) the yield is improved by optimizing the proton transfer system. Based on this, the author's research provides a good reference for the transformation of other P450 oxidase.
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