Structural and biochemical insight into allosteric regulation of the human 2-aminoadipic semialdehyde synthase, a bifunctional enzyme involved in lysine catabolism.

Multiple clinically significant inborn errors of metabolism occur in the lysine degradation, even so the regulation of this biochemical pathway remains understudied. The initial rate-limiting step of lysine catabolism is catalyzed by the bifunctional enzyme 2-aminoadipic semialdehyde synthase (AASS). Therefore, understanding the regulation of AASS activity in normal and disease states will be critical for developing novel therapeutic approaches that modulate lysine degradation flux. Here, we report the cryo-EM structure of the full-length 400 kDa tetrameric AASS enzyme complex with substrates and products bound to both the lysine-2-oxoglutarate (LOR) and saccharopine dehydrogenase (SDH) catalytic domains. Our full-length structure shows that two SDH dimers are connected by a long alpha-helix group and flexible loops to a core LOR tetramer. This functional arrangement gives the SDH domain flexibility to move and readily access the products from the LOR domain. Through chemical screens, we also identified allosteric compounds that modulate activity of the AASS. Maleimides, a class of Michael-Addition chemistry substrates, activate the enzyme by reacting with cysteine 414 and shifting the pH optimum for biochemical activity. This observation can be explained mechanistically by pronounced changes in the affinity for NADPH as a function of pH. Remarkably, the mutagenesis of this allosteric Cys414 has dramatic effects on the enzyme's conformation and activity despite being more than 12 Å from the active site. Simulations also show how changes at this distal site can affect the dynamics of the enzyme. Another compound, 1-methylnicotinamide, inhibits the enzyme by catalyzing the formation of a disulfide bond, which was observed through cryo-EM structural studies. Combined, our data indicate that AASS can be modulated at multiple allosteric sites through small molecules providing opportunities to regulate lysine catabolism by either inhibition or activation. Moreover, small changes near the hinge of an enzyme can have large effects on the catalytic properties of the enzyme. Lastly, targeting allosteric cysteines may be a general strategy for modulating enzyme activity by altering the dynamics of the enzyme.