Structure and Function of N-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 N-acetylmannosamine kinase (NanK) catalyzes the phosphorylation of N-acetylmannosamine to N-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 Fusobacterium nucleatum (Fn-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 Fn-NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria Haemophilus influenzae (Hi-NanK), Pasteurella multocida (Pm-NanK), and Vibrio cholerae (Vc-NanK). Our studies show a 10-fold decrease in substrate binding affinity between Fn-NanK (apparent KM ≈ 700 µM) and ZnF motif-containing NanKs (apparent KM ≈ 60 µM). To understand the structural features that combat the loss of the ZnF motif in Fn-NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from P. multocida and H. influenzae. Here, we report Pm-NanK:unliganded, Pm-NanK:AMPPNP, Pm-NanK:ManNAc, Hi-NanK:ManNAc, and Hi-NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of Fn-NanK with Hi-NanK, Pm-NanK, and hMNK (human N-acetylmannosamine kinase domain of UDP-N-acetylglucosamine-2-epimerase/N-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 Fn-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 Fn-NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.

Molecular characterization of the interaction of sialic acid with the periplasmic binding protein from Haemophilus ducreyi.

The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host-derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi The structure of Hd-SatA in the native form and sialic acid-bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site.

Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.