Mechanistic Insights into Cyclodipeptide Formation by Cyclodipeptide Synthases: A Preliminary Exploration on Pathways and Catalytic Residues

Cyclodipeptide synthases (CDPSs) are enzymes that synthesize cyclodipeptides using two aminoacyl-tRNAs as substrates, but their mechanism remains unclear. This study aims to elucidate the mechanism of AlbC, a CDPS that produces cyclo(L-Phe-L-Phe). We employed small-model quantum mechanics (QM) calculations to propose an intrinsic pathway and molecular dynamics (MD) simulations to identify key catalytic residues involved in this process. The mechanism involves three main steps: activation of Ser37 and the first tRNAPhe to form a Phe-enzyme intermediate, binding of the second tRNAPhe to form a dipeptidyl enzyme intermediate, and intramolecular cyclization to yield the cyclodipeptide. Our QM calculations suggest that Ser37 can be activated through direct transfer of its hydroxyl proton to the O3’ atom of the first substrate. MD simulations highlight the roles of Gly35, Asn40, and His203 in stabilizing the Phe-enzyme intermediate, thus lowering the calculated intrinsic barrier. In the second step, the dipeptidyl enzyme intermediate is favored over the nucleoside intermediate and is stabilized by Asn40, Gln182, and His203 in the AlbC active site, where Gln182 may act as a catalytic base. Additionally, Asn159 and His203 contribute to lowering the significant energy barrier observed in QM calculations for intramolecular cyclization, with Glu182 potentially serving as a catalytic base during this process. Overall, our results support the roles of Asn40 and His203 throughout all mechanistic steps, while highlighting Glu182's involvement in the formation of the dipeptidyl enzyme intermediate and intramolecular cyclization steps. These insights can guide future enzymatic modeling studies of AlbC and potentially other CDPS enzymes using similar approaches.