A cryptic aromatic binding pocket in the highly thermostable GpgS from Mycobacterium hassiacum hints at MGLP pathway...

Mycobacteria have long posed a threat to human health, being responsible for a range of hard-to-treat infections such as tuberculosis, leprosy, and other chronic diseases caused by nontuberculous mycobacteria. Their intrinsic resistance to antibiotics and resilience when exposed to environmental stress complicate treatment and underscore the need for discovering novel drug targets and developing new therapeutic strategies. Mycobacterium hassiacum, a thermophilic mycobacterium, is a promising source of stable proteins suitable for detailed structural analysis. In this study, we investigated its glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme that plays a key role in the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis and is implicated in responses to nitrogen starvation and thermal stress in different mycobacterial species. We confirmed the potential of M. hassiacum as a source of enzymes amenable to structural studies, as its GpgS ortholog yielded not only the first mycobacterial GpgS crystallographic structure at near-atomic resolution (1.10 Å) but also multiple high-resolution structures under different pH conditions and in complex with various substrates. These structural insights revealed a previously unrecognized aromatic binding pocket that is not only cryptic in nature but also capable of accommodating the substrates p-aminobenzoic acid and p-hydroxybenzaldehyde, which have been found to be relevant in its genetic context, thereby establishing a direct link between GpgS activity and broader metabolic pathways in mycobacteria. Our findings establish M. hassiacum as a valuable model for structural enzymology in mycobacteria and highlight GpgS as a potential drug target. The detailed structural data provided here offer a foundation for rational drug design, opening new avenues for the development of inhibitors targeting this key, potentially pleiotropic enzyme.