Deciphering the bidirectional catalytic mechanism of HGPRT from Mycobacterium tuberculosis: Functional mapping of key active-site residues

As the number of tuberculosis cases worldwide has increased, the enzyme hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis ( Mt HGPRT) has emerged as a promising target for drug development because of its role in purine salvage and nucleotide homeostasis. However, the bidirectional catalytic mechanism remains poorly understood, posing challenges for rational drug design. Here, residue-specific functional mapping via systematic mutagenesis coupled with molecular dynamics reveals key residues governing the forward and reverse catalysis. D123 stabilizes α-D-5-phosphoribosyl-1-pyrophosphate binding in the forward reaction, whereas V124 and K154 enhance purine ring stability in the reverse reaction. In addition, D126, V176, L181, and D182 regulate substrate coordination and active site conformation. Cross-species validation confirmed the conserved roles of D123 and V124. These findings provide a structural blueprint for enhancing our understanding of the catalytic mechanism of Mt HGPRT and offer insights into drug design targeting HGPRT-related diseases and anti-tuberculosis therapies.