N-acetyl sugar amidotransferase (NASAT) is involved in the lipopolysaccharide (LPS) biosynthesis pathway that catalyzes the formation of the acetamido moiety (sugar-NC(=NH)CH3) on the O-chain. So far, little is known about its structural and functional properties. Here, we report the crystal structure of an N-acetyl sugar amidotransferase from Legionella pneumophila (LpNASAT) at 2.33 Å resolution. LpNASAT folds into a compact basin-shaped architecture with an unusually wide and open putative substrate-binding pocket and a conserved zinc ion-binding tetracysteine motif. The pocket contains a Rossmann-like fold with a PP-loop, suggesting that the NASAT-catalyzed amidotransfer reaction probably requires the conversion of ATP to AMP and PPi. Our data provide structural insights into the NASAT family of proteins, and allow us to possibly identify its functionally important regions.

Legionella pneumophila , the causative agent of Legionnaires’ disease, utilizes a type IV secretion system (T4SS) to translocate effectors into host cells, modulating diverse cellular processes to create a replication-permissive niche. Here, we characterize Ceg10, a T4SS-translocated effector, as a nucleus-targeting acetyltransferase that interferes with host ribosome biogenesis and cell cycle progression. Structural analysis reveals that Ceg10 harbors a conserved Cys-His-Asp (CHD) catalytic triad required for its acetyltransferase activity. Upon nuclear import mediated by the host transport adaptor HEATR3, Ceg10 selectively acetylates the ribosomal protein RPS20 at Thr64, Thr65, and Arg66, which have not been annotated as posttranslational modification sites. This acetylation impairs RPS20’s interaction with RPS29 and 18S rRNA, two components critical for 40S ribosomal subunit assembly, leading to translation inhibition and G1/S cell cycle arrest. These host perturbations are essential for efficient early-stage intracellular replication of L. pneumophila . Our findings identify a distinct mechanism by which a bacterial effector co-opts nuclear import machinery and directly modifies ribosomal proteins to subvert host biosynthesis and cell cycle control, highlighting ribosomal protein acetylation as a hitherto unrecognized role in host–pathogen interactions.

Members of the YafK subfamily within the YkuD superfamily have been redefined as cysteine-dependent amide hydrolases involved in cell envelope remodeling. However, whether this conserved scaffold supports additional catalytic chemistries remains unclear. Here, we report the structural and functional characterization of Lpg1514 from Legionella pneumophila. Crystal structures show that Lpg1514 adopts a canonical YafK-like fold with a well-defined substrate-binding pocket. Biochemical analysis reveal that Lpg1514 mediates β-lactam turnover. Unexpectedly, this activity depends on a conserved serine residue (Ser132), whereas the canonical cysteine (Cys174) is not required for catalysis under the conditions tested. Mutational and kinetic analysis support a noncanonical serine-dependent mechanism distinct from both YafK-type amidases and cysteine-dependent L,D-transpeptidases. Functionally, Lpg1514 contributes to β-lactam tolerance in L. pneumophila in a Ser132-dependent manner, consistent with limited catalytic efficiency and a role in transient antibiotic interaction rather than productive hydrolysis. Together, these findings expand the functional repertoire of the YafK scaffold and demonstrate that this fold can accommodate mechanistically distinct chemistries. The emergence of serine-dependent β-lactam reactivity within a cysteine-centered lineage highlights the catalytic plasticity of the YkuD/YafK family and suggests a potential route for the diversification of antibiotic-interacting functions.