Abstract
Mycobacterial inorganic pyrophosphatase (Mt-PPa) plays an essential role in Mycobacterium tuberculosis survival both in vitro and in vivo. This enzyme family hydrolyzes inorganic pyrophosphate (PPi) to release inorganic phosphate (Pi), thereby preventing pyrophosphate toxicity. The M. tuberculosis gene Rv3628 encodes a type I inorganic pyrophosphatase that exhibits metal-ion-dependent catalytic activity. In addition to its canonical pyrophosphatase function, Rv3628 also shows the ability to bind nucleotide triphosphates (GTP/ATP). The interaction between GTP and Rv3628, evaluated using isothermal titration calorimetry, revealed mild yet meaningful GTP-binding activity. Because GTP functions as a secondary messenger involved in numerous mycobacterial metabolic pathways, this interaction may represent an important component of cellular signaling cascades.
This study adopts a trilateral strategy integrating computational analysis, mutational profiling, and virtual screening to identify potential druggable features of Mt-PPa. Computational analyses indicated extensive interactions between Mt-PPa and genes associated with GTP- and ATP-dependent pathways. Epitope mapping further identified multiple amino acid regions within PPa that could act as immunologically relevant epitopes. Additionally, Mt-PPa was found to be sensitive to phosphorylation, with several Ser/Thr/Tyr residues serving as putative kinase targets.
Mutational analysis identified W102G, V150G, F44G, I119G, L93F, F3G, F122G, I108G, L32G, M82G, Y17G, L59G, V5G, V26G, I7G, W140D, W140G, W140A, F80G, W140S, L49G, L56G, I9G, V60G, V19G, V92G, L28G, L61G, Y126E, and F123G as the top 30 destabilizing or functionally significant mutations. Among active-site residues, Y126G, Y42G, R30G, E8G, and K16G emerged as the most impactful mutational hits. Enzyme activity was also highly sensitive to temperature and pH, and mutations at His21 and His86 were found to shift the optimal pH range.
Virtual screening of 700 natural compounds from the Herbal Ingredient Targets (HITs) subset of the ZINC database identified ZINC000003979028, ZINC000003870413, ZINC000003870412, ZINC000150338758, ZINC0000070450948, ZINC000150338754, ZINC000095098891, ZINC000000119985, and ZINC000005085286 as top predicted binders to Mt-PPa. Additionally, the known GTPase inhibitors Mac0182344 and NAV-2729 were identified as promising molecules capable of targeting and inhibiting Mt-PPa activity.
Keywords
Mycobacterium tuberculosis, PPa, GTPase inhibitor, GTP, ITC