Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts its in vivo function and the survival of the pathogen

N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmU_Mt) has features, such as a C-terminal extension, that are not present in GlmU_orthologs from other bacteria. Here, we set out to determine the uniqueness of GlmU_Mt by performing in vivo complementation experiments using RvΔglmU mutant. We found that any deletion of the carboxy-terminal extension region of GlmU_Mt abolishes its ability to complement the function of GlmU_Mt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmU_Mt. Furthermore, the co-expression of GlmU_Mt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmU_Mt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmU_Mt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmU_Mt participates in unique interactions essential for its in vivo function.