Conformational Remodeling Underlies Activity Loss in Disease-Linked Asparagine Synthetase Variant
Asparagine synthetase deficiency (ASNSD) is a devastating congenital disorder characterized by profound neurological impairment and early childhood mortality. It is associated with pathogenic mutations in the asparagine synthetase (ASNS) gene. Despite the critical role of ASNS in the amino acid cycle, the molecular basis by which ASNSD-linked missense mutations impair enzyme function remains poorly understood. Here, we present a comprehensive characterization of a recurrent ASNSD-linked variant, R48Q. Steady-state kinetic assays reveal severe reductions in L-glutamine-dependent catalysis and disrupted product stoichiometry, implicating impaired interdomain communication. Cryogenic electron microscopy (cryo-EM) and 3D variable analysis of the EM map uncovers altered loop conformations at the N-terminal active site and subtle conformational changes at the C-terminal domain. Consistent with the structural data, molecular dynamics simulations support that the local disruption propagates across the protein, thereby decoupling coordinated domain motions essential for catalysis. Additionally, we demonstrate that the flanking arginine and the affected loop are evolutionarily conserved across Class II glutamine amidotransferases, highlighting their shared mechanistic importance. These findings provide the molecular basis of an ASNSD variant and establish a framework for understanding how point mutations disrupt complex enzyme dynamics, with broad implications for precision medicine. SignificanceUnderstanding how mutations affect multidomain enzymes is crucial for elucidating the molecular mechanisms underlying genetic disorders. Here, we examine the molecular consequences of the R48Q variant in human asparagine synthetase (ASNS), the sole enzyme responsible for de novo L-asparagine synthesis; mutations of this enzyme lead to a fatal neurometabolic disorder, asparagine synthetase deficiency (ASNSD). By combining biochemical, cryogenic electron microscopy, and molecular dynamics simulation, we show that a single N-terminal amino acid substitution disrupts both local and global coordination, impairing enzyme activity. Our work provides the first mechanistic blueprint of an ASNSD-linked variant. These findings not only deepen our understanding of ASNS but also offer a generalized framework for studying the dynamic regulation of multidomain enzymes in disease.