ABSTRACT
The Rauvolfia serpentina strictosidine synthase (RsSTR) enzyme with a bisubstrate activity is central to monoterpenoid indole alkaloid (MIA) biosynthesis pathways, as it stereoselectively condenses the terpenoid and indole metabolites, secologanin and tryptamine, respectively, into strictosidine. Here, cooperativity was aimed to be deciphered by proxy with help of a non-substrate tryptamine analog (decoy compound) to allow a bisubstrate binding without reaction, facilitating an isothermal titration calorimetry (ITC)-based analysis of the effect of the presence of one substrate on the binding of the other. Tryptamine and tryptamine analog bound to RsSTR with similar binding affinities (Kd). On the contrary, ITC revealed an exothermic titration of secologanin to RsSTR but could not fully quantify it because of weak binding. Interestingly, secologanin bound to RsSTR with an apparent binding affinity (Kd,app) of 212.1 µM in the presence of the decoy compound, as opposed to a lack of binding to RsSTR alone, strongly suggesting a "tryptamine-first" mode of binding. Conversely, binding of tryptamine analog in the presence of secologanin was enhanced >3-fold. Further, molecular dynamics simulation (MDS) analyses revealed the conformational flexibility needed for such cooperativity. Our binding studies complemented with the computational analyses suggested cooperativity in the ordered bisubstrate binding to RsSTR. Therefore, understanding thermodynamics and cooperativity in the binding of substrates or ligands would help to unravel the mechanism of enzyme catalysis and ligand-receptor interactions, and would guide the redesign of enzymes for enhanced properties and the design of inhibitors against enzymes and receptors.Communicated by Ramaswamy H. Sarma.
