Conformational Remodeling Underlies Activity Loss in Disease-Linked Asparagine Synthetase Variant

Understanding 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.