Structural and Biochemical Characterization of a Minimal Protein‐Asparaginase

Enzymatic deamidation of proteins, catalyzed by protein glutaminase (PG) for Gln or by protein asparaginase (PA) for Asn residues, is a key strategy for improving functional properties such as solubility and foaming. However, the only known PA, from Luteimicrobium album (LalPA), is a large, thermally unstable multidomain protein (1355 aa) that has proven challenging to express heterologously. To overcome these limitations, we identified a novel, compact PA from Amycolatopsis deserti (AdePA) using a comprehensive database search. We then solved the first experimental structure of any PA, which revealed a catalytic mechanism utilizing a Ser-His-Asp catalytic triad indicative of a serine protease-like function, which is distinct from that of L-asparaginase. AdePA offers significant advantages over LalPA; it is a smaller (785 aa) single-domain enzyme with superior thermal stability (retaining 50% activity at 40°C, where LalPA is inactivated) and is readily produced through heterologous expression. Furthermore, AdePA shows inverted substrate specificity, preferring sterically small N-terminal groups, making it highly effective for modifying unstructured proteins like gelatin. These findings demonstrate that AdePA is a robust candidate for industrial applications in protein modification.