6q28

Publication Abstract from PubMed

In environments where glucose is limited, some pathogenic bacteria metabolise host derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, Open-reading frame, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding ExGH motif is altered to ExGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterisation of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog N-acetylglucosamine-bound crystal structures (2.33, 2.20 and 2.20 A resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the ExGY motif reveals that the tyrosine binds to the N-acetyl group to select for the 'boat' conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterisation provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.,Coombes D, Davies JS, Newton-Vesty MC, Horne CR, Setty TG, Subramanian R, Moir JWB, Friemann R, Panjikar S, Griffin MDW, North RA, Dobson RCJ J Biol Chem. 2020 Jan 15. pii: RA119.010526. doi: 10.1074/jbc.RA119.010526. PMID:31949045^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.