University of Tokyo and Tohoku University Researchers Visualize SacC
Researchers at the University of Tokyo and Tohoku University have captured near-atomic resolution images of SacC, a bacterial enzyme that assembles the five-ring core of safracin B in a single pass. The discovery, published in the Journal of the American Chemical Society, provides an approach with potential applicability to a broad range of related enzymes that could open new avenues for research into optimizing already existing compounds and creating new ones.
Single-Pass Catalysis of the Safracin B Scaffold
The production line for the soft tissue sarcoma drug trabectedin relies on safracin B. Inside bacteria, the SacC enzyme executes a seven-step catalytic cascade. It stitches together two new carbon-to-carbon bonds and builds a complex five-ring core scaffold in water at room temperature. Artificial synthesis of such a scaffold typically requires multiple carefully staged steps and strict control over conditions. Capturing this assembly process has long challenged structural biologists.
Enzymes in this family function like a factory assembly line. As they pass an unfinished molecule from station to station, their internal parts swing around significantly. This motion causes molecules to freeze in varied poses during sample preparation, resulting in blurred averages. Furthermore, the enzyme tends to clump together in solution.
Overcoming Aggregation with the SacE Partner Protein
To solve the dual problems of molecular motion and sample aggregation, the research team combined chemical and structural biology approaches. They designed and synthesized small molecules that imitate the unfinished intermediates produced during the enzyme’s cascade reaction. To stabilize the enzyme and prevent clumping, the team identified a small partner protein.
As Ryo Tanifuji recalled, For a long time, the project seemed impossible. The enzyme simply would not behave in solution, and without a well-dispersed sample there was nothing to image.
The turning point arrived when the researchers found SacE, a small partner protein coded within the same stretch of bacterial genome. This protein dramatically improved the behavior of the enzyme in solution.
Cryo-Electron Microscopy Analysis at Tohoku University
With the sample stabilized, the scientists flash-froze the preparations in a thin layer of ice. They imaged them using a cryo-electron microscope at Tohoku University. Reconstructing thousands of individual enzyme images yielded a three-dimensional model with a near-atomic resolution.
Our structure captures an early moment in the enzyme cycle, the point where the building block has just been loaded. The later steps, where the rings are closed and the finished product is released, are still out of focus. Capturing those states is our next target, and we expect it will take both better probes and new ways of sorting the images,
noted Ryo Tanifuji.
The research group was led by Ryo Tanifuji and Hiroki Oguri from the University of Tokyo alongside Yoshikazu Tanaka from Tohoku University. Their findings appeared in the Journal of the American Chemical Society under DOI: 10.1021/jacs.6c09555.
