A structure-based mechanism of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (MpaCobU) from Methylocapsa palsarum

Abstract

Adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) is one of the key enzymes that participate in the biosynthesis of cobalamin, specifically lining the lower ligand 5,6-dimethylbenzimidazole in the α-position of cyclic tetrapyrrolidine. During this process, CobU exhibits two distinct activities: kinase and nucleotidyl transferase, using two nucleoside triphosphates. A structural study of CobU from Salmonella typhimurium showed that guanosine triphosphate binding induces a conformational rearrangement of helix 2. This rearrangement decreases the distance between the phosphate binding loop (P-loop) and helix 2, which is important for the subsequent guanylylation step of the reaction. However, these findings provide only partial insights into the mechanism of CobU at the structural level, and the precise molecular details of this mechanism have not yet been studied. As a first step towards elucidating the molecular mechanisms and sequence of events involved in the phosphorylation and guanylylation steps, we report the high-resolution crystal structures of phosphorylated -MpaCobU (1.8 A), the C91S mutant (1.5 A), the guanosine diphosphate complex (1.9 A), and the adenosylcobinamide-phosphate complex (2.6 A) from Methylocapsa palsarum for the first time. High-resolution structures revealed the crucial elements governing the catalytic steps of MpaCobU, thereby contributing to understanding the catalytic mechanism of CobU at the molecular level. ⓒ 2024 The Authors