How a metabolite protects the enzyme that consumes it

Metabolites are consumed by enzymes to produce other vital molecules as well as to control metabolite levels. But how do cells avoid consuming too much? Researchers from the MPI of Biochemistry, with the University of Nevada, Las Vegas, and the de Duve Institute in Brussels, have deciphered such molecular regulation in tryptophan metabolism. The essential amino acid tryptophan binds to a second site on the enzyme TDO2, distinct from where it is consumed. This sequesters a region needed by two effectors, the kinase CK2 and the ubiquitin ligase CUL1–β-TrCP, which act sequentially to signal TDO2 degradation. The findings are now published in Molecular Cell.

TDO2—a key enzyme in tryptophan metabolism

The essential amino acid tryptophan is a key building block for proteins and also serves as a precursor for serotonin, melatonin, niacin (vitamin B3) and NAD+. These molecules are vital for the nervous and immune systems and the cell's energy supply. As tryptophan can only be obtained through dietary supply, the body must maintain tryptophan levels within a physiological range.

The enzyme tryptophan 2,3-dioxygenase (TDO2) carries out the first and rate-limiting step of the kynurenine pathway. While the cell requires the downstream products of TDO2, it must also avoid consuming too much tryptophan.

Researchers have known since the 1950s that TDO2 is stable when there is sufficient tryptophan and is rapidly broken down when tryptophan supply is limiting. It has recently been shown that this feedback mechanism relies on the ubiquitin-proteasome system, the cellular system that marks and degrades unwanted proteins. How tryptophan controls this system has now been elucidated.

Two steps controlling TDO2 degradation

Alina Thielen, the first author of the study, explains, "To identify the molecular mechanism underlying TDO2 degradation, we conducted a genome-wide CRISPRi screen. This enabled us to discover the two proteins controlling the degradation process: the kinase CK2 and the E3 ligase CUL1–β-TrCP. To understand their mechanism of action, we performed biochemical reactions with purified proteins in a test tube and used cryo-electron microscopy to examine the structure of TDO2 with and without tryptophan occupying the second binding site."

The kinase and E3 ligase proteins act sequentially on TDO2 to mark it for degradation. When tryptophan becomes scarce, the kinase CK2 attaches phosphate groups to TDO2. This phosphorylation serves as a recognition signal for the E3 ligase CUL1–β-TrCP, which then attaches ubiquitin to TDO2. Ubiquitin-tagged TDO2 is recognized by the proteasome, the cell's waste disposal system.

Thielen explains, "It was already known that tryptophan binds to a second site on TDO2, which is located distal from the catalytic center. Using cryo-electron microscopy, we were able to see how tryptophan organizes a section of the protein near this second binding site to stabilize TDO2. This section folds over the bound tryptophan like a flap, so that the nearby recognition sequence for CK2 and CUL1–β-TrCP is inaccessible.

"If the tryptophan level drops, tryptophan dissociates from the second binding site. As a result, the flap becomes unstructured, making the recognition sequence for CK2 and CUL1–β-TrCP accessible and thereby initiating the process of protein degradation."

Therapeutic relevance of the TDO2 stabilization mechanism

Upregulation of TDO2 has been observed in neurological disorders and various types of cancer. In tumors, elevated TDO2 levels can weaken the antitumor immune response. TDO2 is therefore a potential target for new therapeutic agents.

Brenda Schulman explains, "A detailed understanding of TDO2 regulation is important to be able to utilize TDO2 as a therapeutic target. A drug that targets the site where the enzyme consumes tryptophan could also bind to the second binding site and thereby promote unintended TDO2 stabilization. On the other hand, a molecule that occupies the second binding site but keeps the recognition sequence for CK2 and CUL1–β-TrCP accessible could induce the degradation of TDO2."

Schulman summarizes the new findings: "Tryptophan is not only a metabolite and substrate for TDO2—it also acts as a signal that determines how long the TDO2 enzyme remains stable within the cell. Our study reveals a general principle for how a metabolite directly controls the stability of the enzyme that consumes it, thereby linking the cell's metabolism to protein degradation. We are now investigating how cells use this strategy more widely to regulate metabolism according to nutrient supply."