Structural basis for allosteric regulation of mycobacterial guanosine 5´-monophosphate reductase by ATP and GTP

Guanosine 5′-monophosphate reductase (GMPR) is a crucial enzyme in purine salvage pathway that catalyzes the NADPH-dependent conversion of GMP to IMP, thereby contributing to purine nucleotide homeostasis. Mycobacterium smegmatis GMPR ( Msm GMPR) contains a regulatory cystathionine β-synthase (CBS) domain, which mediates allosteric modulation by ATP and GTP. However, Msm GMPR exhibits an atypical tertiary structure that is incompatible with the acknowledged regulatory mechanisms of IMPDH/GMPR family enzymes. Here, we combine X-ray crystallography, cryo-electron microscopy, and biochemical binding assays to elucidate the molecular basis of Msm GMPR regulation by ATP and GTP. We show that ATP stabilizes a compressed conformation that inhibits the enzyme by restricting access to the active site and preventing NADPH binding. In contrast, GTP counteracts ATP binding, promoting an active conformation that enables catalysis. Our results provide insight into how Msm GMPR senses and responds to the cellular purine nucleotide balance, revealing a novel mode of allosteric regulation by a CBS domain.