Biocatalytic synthesis of S ‐adenosyl‐ l ‐methionine analogues for allosteric activation of cystathionine‐β‐synthase

Cystathionine‐β‐synthase (CBS) is the key regulatory enzyme of the transsulfuration pathway and plays a crucial role in eliminating homocysteine. Homocysteine, a by‐product of methylation reactions, is a known risk factor for cardiovascular, neurodegenerative and metabolic disorders, all of which are among the leading causes of death worldwide. CBS represents a promising therapeutic target due to its allosterically inducible capacity to redirect homocysteine into sulfur metabolism, yielding the protective antioxidants cysteine, hydrogen sulfide and glutathione. Although the therapeutic benefit of CBS activation was established more than a decade ago, there are as yet no drug‐like small‐molecule allosteric activators of CBS. Therefore, we establish a framework for the development of CBS activators. Starting from S ‐adenosyl‐

‐methionine (SAM), the endogenous allosteric activator of CBS as a template, we rationally designed 15 analogues and synthesized them using biocatalysis. We characterized their action on isolated recombinant human CBS and cell lysate and their metabolic breakdown in DNA methylation. Besides SAM, three analogues enhanced the catalytic activity of CBS and stabilized CBS during thermal denaturation, suggesting binding. Thermodynamic parameters were quantified using isothermal titration calorimetry (ITC). All active compounds participated in the methylation of lambda DNA. We confirmed S ‐adenosyl‐ ‐ethionine, an established CBS activator, and seleno‐adenosyl‐ ‐methionine, recently reported by us. Additionally, we introduce a novel CBS activator, S ‐2‐amino‐adenosyl‐ ‐methionine. Taken together, CBS allosteric activation required compounds that do not deviate far from the structure of SAM. Nevertheless, we discovered a new allosteric activator of CBS and propose that our work represents a structural framework for further development of small‐molecule activators of CBS.