Substrate-Induced Dynamic Regulation of the Catalytic Loop in Assisting Allosteric Communication in Formylglycinamidine Synthetase

Bifunctional enzymes that execute tandem chemical reactions progress through orchestrated conformational states to achieve chemical synchronization. In these allosterically regulated systems, specific stimuli, such as substrate and cofactor binding, determine reactivity. Here, we employ a combination of steady-state and time-resolved fluorescence methods to monitor the conformational dynamics of a catalytic loop in formylglycinamidine synthetase, an enzyme that catalyzes a crucial step toward the synthesis of precursors of DNA and RNA. We show that the catalytic loop harbors adaptive structural elements that change secondary structure in response to substrate binding and, thereby, enable allosteric cues to the 25 Å distal NH₃-producing site. To exclusively track the conformational changes in the loop, a fluorescent unnatural amino acid was introduced into the 1300-amino acid protein, allowing for a unique signal that was not masked by the indigenous fluorescent amino acids. The study highlights the role of flexible small elements that act as triggers of the allosteric cycle and maps states that are essential for function.