Shanghai Jiaotong University Deng Zixin's team reveals the catalytic cycle trajectory of dimerized non-ribosomal peptide synthases

Recently, the team of Professor Zixin Deng from the School of Life Science and Technology of Shanghai Jiao Tong University and the State Key Laboratory of Microbial Metabolism published in the journal Nature Communications entitled "Epimerase-embedded dimerization of non-ribosomal peptide synthases.
"Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain" research paper
.

By combining structural biology and biochemistry, this work analyzes the three-dimensional structure of multiple catalytic states of the pathogenic factor synthesis machine, captures its continuous catalytic cycle trajectory, and further promotes the structural study of secondary metabolic giant synthases.

Non-ribosomal peptide synthetases (NRPSs) are a class of multi-domain, modular giant synthases capable of synthesizing natural products with a wide range of biological activities, ranging from clinical drugs (antibiotics, anti-tumor inhibitors, and immunosuppressants) to pathogenic drugs.
Disease factors (carcinogenic molecules, siderophores, etc.
)
.

NRPSs catalyze in an "assembly-line" fashion, in which the extension module catalyzes the following steps: substrate activation, intermediate product shuttling, and condensation reactions; sometimes, modified epimerization reactions

However, the lack of high-resolution structural information on the extended modules of NRPSs hinders further understanding of the catalytic mechanism of these giant synthases
.

For example, how do key domains and amino acids in NRPSs perform substrate recognition, activation and extension? How is the organization of the modifying domain embedded in the holoenzyme? How does an intact giant synthase perform end-product synthesis by shifting key domains? How do the various domains of itself dynamically coordinate to complete the catalytic cycle?

In response to the above-mentioned key issues, the team of Prof.
Zixin Deng successfully resolved three high-resolution conformations of a typical NRPS extension module PchE at 2.
97 Å, 3.
78 Å and 3.
47 Å through cryo-electron microscopy single particle analysis (Fig.
1)
.

The giant synthase is responsible for the biosynthesis of the pathogenic factor Pyochelin in Pseudomonas aeruginosa, and contains five catalytic domains: aryl carrier protein (ArCP), condensed cyclization domain (Cy), adenylation structure domain (A), epimerase (E) and peptidyl carrier protein (PCP)

Figure 1.
Framework of the three conformations of PchE

The three conformations of PchE correspond to three different catalytic states: substrate supply, condensation and post-condensation
.

The separate capture of the three clarified the binding mode of the Cy domain to the two carrier proteins; established the interaction relationship between the substrate, the final product, the substrate channel, and the active site (Figure 2)

Figure 2.
Substrate supply, condensation and post-condensation catalytic state of Cy domains

Using the means of neural network deep learning, the researchers further observed the continuous dynamic trajectory of the complete catalytic cycle of PchE, from substrate activation to epimerization reaction (Fig.
3, Video 2)
.

The catalytic reaction starts in conformation (i), with ArCP bound to the Cy domain, representing the substrate-donating conformation

Figure 3.
Catalytic cycle trajectory of PchE

In summary, the typical NRPS extension module PchE drives the synthesis of the final product by integrating structural rearrangements in multiple dimensions, like a well-designed dance of biological macromolecules
.

The large-scale movement of the domains creates different catalytic states, the switch of the substrate channel controls the entry of small molecule compounds, and the positions of key amino acids coordinate the progress of the catalytic reaction

Tristan Croll, associate researcher of Cambridge University Institute of Medical Research (CIMR), Cao Wei, a doctoral student from Shanghai Jiaotong University, Chen Lu, a master student, Zhang Wei from the instrument platform of the Academy of Biological Sciences, and Kong Liangliang from the National Center for Protein Science (Shanghai) participated in the work
.

Thanks to Wang Fangfang, Xin Lihui, Li Guangyi, Duan Jialin, Liu Na and other teachers of the National Protein Center for their technical support
.

Thanks to the π 2.

This research was supported by the National Key Basic Research Development Program for Synthetic Biology (2018YFA0900700, 2019YFA0905400, 2021YFA0910500), the National Natural Science Foundation of China (32171252, 91753123, 31470830), the Shanghai Super Postdoctoral Incentive Program (202112) and the Wellcome Trust (209407/Z/ 17/Z) financial support

Video 1 link : https://life.
sjtu.
edu.
cn/Assets/userfiles/files/Supplementary%20Movie%201.
mp4 (F372 controls the opening and closing of the Cy domain substrate channel)

Video 2 link : https://life.
sjtu.
edu.
cn/Assets/userfiles/files/movie2(1).
mp4 (Continuous Dynamic Process of PchE Catalytic Trajectory)

Article link :  https:// class="col-xs-4 control-label">author: