Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum

Abstract

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

Keywords: Fusobacterium nucleatum; N-acetylmannosamine kinase; sialic acid catabolism.

Figure 1

(a) Sialic acid catabolism in F. nucleatum. SiaT, transporter; NanA, lyase; NanK, kinase; NanE, epimerase; NagA, deacetylase; NagB, deaminase. (b) The chemical reaction catalyzed by N-acetylmannosamine kinase.

Figure 2

Overall structure of F. nucleatum apo N-acetylmannosamine kinase. The N-terminal domain is coloured in blue shades, the C-terminal dimerization domain in red shades and the the hinge loops are depicted in yellow. For clarity, based on the human hMNK structure (PDB entry 2yhy), ManNAc (green sticks), ADP (white sticks) and Mg²⁺ (blue sphere) have been modelled in the putative active site.

Figure 3

Overall structures of the N-terminal domain (a) and C-terminal dimerization domain (b). The helices and strands are numbered. The residues that span and flank each domain are marked. Domain 1 starts from the N-terminus and ends at residue 118 and then continues from residue 272 to the C-terminus (blue). Residues 126–268 form the dimerization domain.

Figure 4

FnNanK lacks the cysteine-rich zinc-binding motif. (a) Structural comparison of apo FnNanK in red and substrate-bound (ManNAc, ADP and Mg²⁺) hMNK in green. (b) Superimposition of the substrate-binding regions of bacterial NanKs. The putative residues involved in catalysis in the substrate-binding site in FnNanK (blue) are superimposable with the corresponding residues in NanK from E. coli (EcNanK; PDB entry 2aa4, pink) and L. monocytogenes (LmNanK; PDB entry 4htl, green). The zinc-binding motif is only visible in EcNanK, which is represented by the coordination of Cys173, Cys166, Cys168 and His156 to the Zn atom (grey). The highly conserved histidine that coordinates ManNAc is present in FnNanK and EcNanK but corresponds to a tyrosine in LmNanK.