Structure-based study of CmoA reaction mechanism

Abstract

The 34th uridine of tRNA is almost always modified in living organisms. Uridine is transformed into 5-carboxymethoxyuridine (cmo5U) in Gram-negative bacteria, and the carboxymethyl transferases CmoA and CmoB participate in cmo5U formation. CmoB uses Cx-SAM as the carboxymethyl donor to form cmo5U. Cx-SAM is a product of the CmoA enzymatic reaction, and is a metabolite produced by CmoA using prephenate as a carboxymethyl donor. In this study, I propose the structure of substrate bound to V. fischeri CmoA. In the presented structure, only one analog of the substrates was bound and some structure parts were disordered. The missing part contains Arg199, which specifically binds to Cx-SAM but not SAM in E. coli CmoA. This structure made it possible to speculate that Arg199 can affect the binding affinity between ligand and CmoA depending on the presence or absence of an interacting molecule at the ligand binding site. Binding affinity analysis of E. coli CmoA, V. fischeri, and Cx-SAM was also performed. Finally, ligand was extracted from CmoA, purified under various conditions and analyzed. CmoA enzyme activity was changed according to the location of the histidine tag. When the production of one substrate used for the CmoA enzyme reaction was blocked in the cell, the enzyme reaction did not proceed and it seemed that the binding affinity between the enzyme and the substrate was also changed. Although the exact cause of the difference in enzyme activity depending on the location of the histidine tag has not been identified, it will expand the direction of research by finding one more variable that affects the enzyme activity. Confirmation of the enzymatic reaction according to the presence or absence of a substrate and proof of the mechanism has been done only in vitro so far. However, through the results of this experiment, it was found that changes in the in vivo environment can also lead to differences in enzyme activity. In addition, the result, which is presumed to be a change in binding affinity beyond a change in enzyme activity, can provide structural insight and information on the molecular interaction between the enzyme and the ligand.