Various structures of CmoB provide structural insight into cmo5U modification mechanism

Abstract

The 34th uridine of tRNA is always almost modified in three domains of life. The 5-carboxymethoxyuridiene (cmo5U), which is one of the species of uridine modification, is generated by carboxymethyl transfer enzyme CmoB in gram-negative bacteria. Unique metabolite Carboxy-S-adenosyl-l-methionine (CxSAM) is used for carboxymethyl donor of CmoB reaction and S-adenosyl-l-homocysteine (SAH) is a co-product of the transfer reaction. In this study, I present four X-ray crystal structures of CmoB from Vibrio vulnificus; i.e., apo-, SAH bound, CxSAM bound and tRNA bound forms. The crystal structure of apo-CmoB displays novel open conformation of the protein, which has not been observed in previously reported structures. Flexible loop region is stabilized with neighboring symmetry related molecules. The Crystal structures of CmoB bound with SAH and CxSAM provide molecular basis for cofactor selectivity. In addition, binding assays for SAH with CmoB was conducted to investigate the affinity for the cofactor in solution. Furthermore, a co-crystal structure of tRNA-CmoB complex has been determined for the first time in this study. However, the structure appears to represent an inactive form, where the wobble uridine is in stacked pose and too distant from the bound CxSAM for the transfer reaction to occur. The mutagenesis assay suggests that the amino acid residues Arg-90, Arg-242, Arg-247 and Asn-248 are important to recognizing phosphate backbone of tRNA, validating that the structure provides a valuable information on the molecular interactions between tRNA and the enzyme in forming the pre-Michaelis complex.