Structure and Function of -Acetylmannosamine Kinases from Pathogenic Bacteria.

Several pathogenic bacteria import and catabolize sialic acids as a source of carbon and nitrogen. Within the sialic acid catabolic pathway, the enzyme -acetylmannosamine kinase (NanK) catalyzes the phosphorylation of -acetylmannosamine to -acetylmannosamine-6-phosphate. This kinase belongs to the ROK superfamily of enzymes, which generally contain a conserved zinc-finger (ZnF) motif that is important for their structure and function. Previous structural studies have shown that the ZnF motif is absent in NanK of (-NanK), a Gram-negative bacterium that causes the gum disease gingivitis. However, the effect in loss of the ZnF motif on the kinase activity is unknown. Using kinetic and thermodynamic studies, we have studied the functional properties of -NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria (-NanK), (-NanK), and (-NanK). Our studies show a 10-fold decrease in substrate binding affinity between NanK (apparent ≈ 700 μM) and ZnF motif-containing NanKs (apparent ≈ 60 μM). To understand the structural features that combat the loss of the ZnF motif in -NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from and . Here, we report -NanK:unliganded, -NanK:AMPPNP, -NanK:ManNAc, -NanK:ManNAc, and -NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of -NanK with -NanK, -NanK, and hMNK (human -acetylmannosamine kinase domain of UDP--acetylglucosamine-2-epimerase/-acetylmannosamine kinase, GNE) show that even though there is less sequence identity, they have high degree of structural similarity. Furthermore, our structural analyses highlight that the ZnF motif of -NanK is substituted by a set of hydrophobic residues, which forms a hydrophobic cluster that helps the proper orientation of ManNac in the active site. In summary, ZnF-containing and ZnF-lacking NanK enzymes from different Gram-negative pathogenic bacteria are functionally very similar but differ in their metal requirement. Our structural studies unveil the structural modifications in -NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.