Structural and functional dissection of human neurolysin through alanine‐scanning mutagenesis

Peptidase neurolysin (Nln, EC3.4.24.16) is a zinc-dependent M3 metallopeptidase that regulates key neuropeptide systems, including neurotensin, bradykinin, and substance P. Despite the availability of high-resolution Nln structures, the contribution of individual residues to its catalytic function and structural stability remains incompletely understood. In this study, we systematically examined 24 residues of human Nln using alanine-scanning mutagenesis, steady-state kinetic analysis, and differential scanning fluorimetry (DSF). Mutations were selected to probe residues spanning α helices, flexible loops, and the catalytic channel, categorizing them into two classes: (a) residues critical for structural and conformational stability, and (b) residues involved in substrate recognition and catalysis. Alanine substitutions near the catalytic core (H425, F453, S506, and H601) or within the flexible loop at the base of the catalytic channel (E498 and T499) caused severe or complete loss of enzymatic activity, underscoring their essential roles in substrate binding and active-site geometry. Substitutions disrupting peripheral salt bridges (E73 and R141) predominantly affected thermal stability without markedly altering catalytic efficiency. Conversely, variants K145A and R491A retained activity with potential trends toward enhanced turnover, identifying these regions as permissive sites that may support gain-of-function modulation. Collectively, our findings define the residue-level determinants of Nln activity and stability, highlighting how local interactions at the catalytic channel, flexible loops, and key helices govern the conformational dynamics of the enzyme. This work provides a molecular framework for understanding the allosteric pathways in Nln and for designing small-molecule modulators.