The tRNA moieties of both aminoacyl-tRNA substrates of a cyclodipeptide synthase share a common binding site, as revealed by RNA microhelices mimicking tRNA acceptor arms.

Cyclodipeptide synthases (CDPSs) sequentially use two aminoacyl-tRNAs (AA-tRNAs) as substrates to catalyze cyclodipeptide formation. We previously showed that microhelices (miHxs), which mimic the tRNAs acceptor arms, are as efficient as full-length AA-tRNAs as substrates when aminoacylated by flexizymes. We generated a diverse set of miHxs (acylated, unacylated, misacylated, mutated, or shortened miHxs) and analyzed their interactions with CDPSs. We studied the Nocardia brasiliensis CDPS (Nbra-CDPS), which synthesizes cyclo(l-Ala-l-Glu) using Ala-tRNAAla and Glu-tRNAGlu as its first and second substrates, respectively. We determined the crystal structure of Nbra-CDPS bound to two analogues of its first substrate, unacylated miHxAla and acylated miHxAla, in which alanine is attached via an amide bond. We showed by cryoEM that the miHxAla mimics well the acceptor stem of the full-length tRNAAla. We determined the crystal structure of Nbra-CDPS bound to unacylated miHxGlu, an analogue of its second substrate, and showed that, despite sequence differences, it superimposes well with miHxAla. This result, combined with the use of misacylated substrates, indicates that the RNA stem moieties of both substrates share a common binding mode. Together, our findings establish miHxs as powerful tools for dissecting CDPS substrate recognition and provide a framework for studying other AA-tRNA-utilizing enzymes.