Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli

The tRNA modification N6-threonylcarbamoyladenosine (t(6)A) is universally conserved in all organisms. In bacteria, the biosynthesis of t(6)A requires four proteins (TsaBCDE) that catalyze the formation of t(6)A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t(6)A synthesis in vitro with an IC50 value of 1.3 mu M in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

Automated summary

This study investigates the transfer mechanism of the key t(6)A modification in tRNA biosynthesis, focusing on the structure of the TsaBD heterodimer and its interaction with a phosphonate isosteric mimic of TC-AMP. The inhibitor binding to TsaBD via active site Zn atom suggests a potential oxyanion hole for catalysis, highlighting the metal's role as a binding scaffold for the intermediate. The phosphonate-bound crystal structure provides insights for the rational design of potent small molecule inhibitors for potential antibiotics.