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The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus

Coombes, D. (författare)
Davies, J. S. (författare)
Newton-Vesty, M. C. (författare)
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Horne, C. R. (författare)
Setty, T. G. (författare)
Subramanian, R. (författare)
Moir, J. W. B. (författare)
Friemann, Rosmarie, 1975 (författare)
Gothenburg University,Göteborgs universitet,CARe - Centrum för antibiotikaresistensforskning,Centre for antibiotic resistance research, CARe
Panjikar, S. (författare)
Griffin, M. D. W. (författare)
North, R. A. (författare)
Dobson, R. C. J. (författare)
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 (creator_code:org_t)
2020
2020
Engelska.
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 295:10, s. 3301-3315
Relaterad länk:
https://gup.ub.gu.se...
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https://doi.org/10.1...
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  • Tidskriftsartikel (refereegranskat)
Abstract Ämnesord
Stäng  
  • In environments where glucose is limited, some pathogenic bacteria metabolize 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, ORF, 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 characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand?free, N-acetylmannosamine?bound and substrate analog GlcNAc?bound crystal structures (2.33, 2.20, and 2.20 ? 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 characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Staphylococcus aureus (S
aureus)
X-ray crystallography
analytical
ultracentrifugation
small-angle X-ray scattering (SAXS)
structural
biology
N-aceytlmannosamine kinase
repressor
open reading frame
kinase
ROK protein family
sialic acid
sugar kinase
x-ray diffraction
sialic-acid catabolism
molecular replacement
escherichia-coli
acetylneuraminate lyase
crystal-structures
bacillus-subtilis
glucose kinase
purification
crystallization
Biochemistry & Molecular Biology

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