The full-length crystal structure of the universal biocatalyst has been cracked

On May 29th Nature-Communication published the first full-length fine 3D structure of the cytochrome P450 enzyme online, solving this decades-old scientific puzzle, Hubei University's School of Life Sciences, the Ministry of The Ministry of the National Key Laboratory of Biocatalytic and Enzyme Engineering Professor Chen Chunqi, Li Lixin and Guo Ruiting completed this workThey parsed the crystal structure of a P450 enzyme, CYP116B46, and clarified how electrons are transmitted within moleculesThis provides important guidance for understanding the structure and complete mechanism of P450 and of great value to the application of P450 enzyme in the pharmaceutical and industrial fieldsuniversal biocatalystcytochrome P450 is an enzyme protein first found in liver cells in mice in 1955, named for its reduced-state absorption spectrum wavelength associated with carbon monoxide at a wavelength of about 450 nmthe protein consists of a hemoglobin (heme) domain and a reductase domain containing lutein mononucleotides (FMN), which are connected by a ferrous protein domain containing a group of iron sulfur"Cytochrome P450 is a large family of enzymes, referred to simply as CYP, that participates in the synthesis pathways of many natural products in response to toxic and drug metabolismChen Chunqi, author of the paper, told The China Science Daily that in plants, the P450 family is involved in the synthesis of specific compounds, such as artemisinin and yewalcohol, and in the human body, the P450 family is mainly involved in detoxification processes, such as the aflatoxin that can cause cancerthis is due to the high catalytic plactability of The P450, involving more than 20 types of reactions, including hydroxyreactation, cyclooxidation reaction and ring reactionIts substrate spectrum is very wide, can identify aromatic, polyketone, steroids, peptides, sugars and other types of substrates, with a "universal biological catalyst" calledit also has a high degree of regional and stereo-selectivity, and can specifically identify structural sites and directionsIn addition, the substrate binding area of the P450 enzyme is highly malleable, and the substrate spectrum can be changed through enzyme engineering, potentially for applications in the manufacture of important chemicals and pharmaceuticalstherefore, drug activity and metabolism are primarily the responsibility of the family of CYP, an enzyme that covers about 75% of such metabolism in organismsThe P450 system is widely present in all living organisms and even viruses, and more than 18,000 P450 systems have been foundMore than 57 P450 systems have been found in the human body, most of which are membrane proteins, mainly distributed in the mitochondrial membrane or the endogenous network However, no trace of such enzyme proteins has been found in E coli "the vast majority of P450 enzymes require redox to provide electrons to activate the hemoglobin in the substrate binding area and thus transform the substrate Chen Chunqi said that building a P450 system must look for homologous or matching redox enzymes only two of the nine families of the P450 system have their own oxidizing enzymes, known as self-sufficient P450 enzymes, in the same peptide chain, which comes mainly from bacteria Other P450 systems require matching redox, which occurs mainly in animals "self-sufficient P450 enzyme is an attractive biocatalyst for biotechnology applications Therefore, how the internal electrons of the self-sufficient P450 enzyme are transmitted is a very interesting and important subject Guo Ruiting, author of the paper, told China Science Daily that scientists had previously been more clear about the study of structural areas such as hemoglobin and FMN in the P450 system, but had always been unaware of the middle connection and lacked a full-length fine structure " In the process of protein purification and crystal culture, The P450 enzyme is very easy to break or degrade from the middle, making it difficult to obtain a complete full-length three-dimensional structure Chen Chunqi said obtained a full-length P450 fine 3D structure
there are currently two types of self-sufficient P450 enzymes known The first category is represented by CYP102A1 from the giant bacillus spore, consisting of the nitrogen-side hemoglobin domain and the carbon-side P450 reductase (CPR), which is transmitted electronically from the carbon side to the nitrogen end Although the full-length fine structure of this P450 enzyme is still missing, there are some rough theories about the electron transfer mechanism of this Type of P450 enzyme second category is CYP116, which is included in the study This type of P450 consists of the heme domain of the nitrogen side and the carbon-side phthalymic diplusase reductase-like tectonosis domain (PDR), which contains the FMN-dependent reductase domain and ferrite protein domain how electrons are transferred from the carbon side to the nitrogen side in a polypeptide chain of self-sufficient P450 enzymes? Because of the chronic lack of full-length protein structure information for more than 30 years, it was not clear before the study Sabine J "Getting a full-length enzyme is a great achievement," said Sabine L Flitsch "Many teams, including her own, have tried to parse these structures because they have significant application potential as biocatalysts " can be said that crystal preparation has transcended science and almost become an art Chen Chunqi said, to obtain a complete large protein crystal structure, the difficulty can be imagined the relative stability of the CYP116B46 used in the study, and the experience accumulated by President Guo Rui in crystal preparation and structural analysis over the years, they were able to analyze their complete crystal structure using X-ray crystallography structural analysis showed that in this enzyme, FMN is close enough to the iron sulfur cluster to achieve direct electron transfer, but hemoglobin is too far from the iron sulfur cluster to allow the electron to transfer directly, but rely on five "second hand" amino acids to achieve transmission " We parsed the crystal structure from the entire length of CYP116B46 and established a clear model of the enzyme structure Guo Said, this continuous peptide folded into three domains, connected by two segments of the linked peptides, from nitrogen to carbon end sequence: hemoglobin binding domain, reductase domain and ferrite protein domain Guo Ruiting introduced, the structure analysis found that the arrangement of cyP116B46 three structural domains and the direction of electronic transmission is very consistent Hemoglobin binding area of the underlying binding area facing outward, is conducive to the bottom into the binding, which shows that the arrangement of the structure is quite reasonable Among them, the straight-line distance between the reductase domain and the ferrite protein domain is 7.9? , is conducive to the direct transmission of electrons " Scientists generally believe that less than 8 ? The distance is that electrons can pass directly past Guo said on the other hand , the linear distance between ferrite protein domain and hemoglobin is 25.3 , beyond the effective direct electronic transmission distance currently recognized 5 "second passer" transmission electron " between the two redox centers, other assistive factors to help the electron transmission is less likely Guo Explained that because the structure shows the narrow passage between the two, there is a lack of other cofactor binding characteristics On the other hand, considering that amino acids are also an electronic carrier , amino acids between the two may also play an important role in the process of electron transmission "Now we have the opportunity to design reductase domains and electronic transfer pathways." Chen Chunqi said To analyze the effects of the side chain groups of these amino acids on the activity of CYPP116B46 enzymes, they mutated these sites into alanicine and measured the amount of product production to evaluate the role of each amino acid Guo Ruiting, the enzyme live test results showed that The activity of the alanine mutants of R388, R718, E723, S726 and E729 decreased significantly By re-calibrating these sites on the structure, you can see the approximate direction of electronic transmission R378A mutant protein lack of red caused by hemoglobin, carbon monoxide treatment after the phenomenon of absorption peak band shift Add in the location of R378 adjacent to hemoglobin, it is speculated that R378 may be related to the binding hemoglobin Therefore, the effect of the mutation of R378A is more complex, and further experiments are needed to determine the effect of this bit on the electron transmission of CYP116B46 " know the role of amino acids in the electron transfer process, we can consider looking for replacement of other effective amino acids to improve the efficiency of electron transfer Guo's explanation is like replacing a longer "second passer" that allows electrons to pass through faster this design will improve the efficiency of the catalytic reaction The analysis of the p450 full-length structure is a very important milestone for understanding the self-sufficient P450 enzyme catalysis mechanism, and it is of great significance for the transformation and application of P450 enzyme (
Bioon.com of Biological Valley)