Probing the determinants of phosphorylated sugar-substrate binding for human sialic acid synthase.

N-acetylneuraminic acid (NeuNAc), the most naturally abundant sialic acid, is incorporated as the terminal residue of mammalian cell surface glycoconjugates and acts as a key facilitator of cellular recognition, adhesion and signalling. Several pathogenic bacteria similarly express NeuNAc on their cell surfaces, allowing evasion of their host's immune system. Prokaryotic NeuNAc biosynthesis proceeds via condensation of phosphoenolpyruvate (PEP) with N-acetylmannosamine (ManNAc), a reaction catalysed by the domain-swapped homodimeric enzyme, N-acetylneuraminic acid synthase (NeuNAcS). Conversely, the mammalian orthologue, N-acetylneuraminic acid 9-phosphate synthase (NeuNAc 9-PS) utilises the phosphorylated substrate N-acetylmannosamine 6-phosphate (ManNAc 6-P) in catalysis. Here we report an investigation into the determinants of substrate specificity of human NeuNAc 9-PS, using model-guided mutagenesis to delineate binding interactions with ManNAc 6-P. Modelling predicts the formation of a domain-swapped homodimer as observed for bacterial variants, which was supported by experimental small angle X-ray scattering. A number of conserved residues which may play key roles in the selection of ManNAc 6-P were identified and substituted for alanine to assess their function. Lys290 and Thr80 were identified as a putative phosphate binding pair, with the cationic lysine residue extending into the active site from the adjacent chain of the dimeric enzyme. Substitution of these residues results in a significant loss of activity and reduced affinity for ManNAc 6-P. These residues, along with the electropositive ß2α2 loop, are likely to facilitate the PEP dependent binding and stabilisation of ManNAc 6-P. By utilising a phosphorylated sugar-substrate, the mammalian enzyme gains considerable catalytic affinity advantage over its bacterial counterpart.

Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase.

N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N-terminal catalytic (ß/α)8 barrel linked to a C-terminal antifreeze protein-like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X-ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen-bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries.

Arg314 is essential for catalysis by N-acetyl neuraminic acid synthase from Neisseria meningitidis.

The sialic acid N-acetylneuraminic acid (NANA) has a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host's immune system. NANA is synthesized in bacteria by the homodimeric enzyme NANA synthase (NANAS), which catalyzes a condensation reaction between phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc). NANAS is closely related to the α-keto acid synthases 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase and 3-deoxy-d-manno-octulosonate 8-phosphate synthase. NANAS differs from these enzymes in that it contains an antifreeze protein like (AFPL) domain, which extends from the C-terminal of the (ß/α)8 barrel containing the active site and contributes a highly conserved arginine (Arg314) into the active site of the opposing monomer chain. We have investigated the role of Arg314 in NmeNANAS through mutagenesis and a combination of kinetic and structural analyses. Using isothermal titration calorimetry and molecular modeling, we have shown that Arg314 is required for the catalytic function of NANAS and that the delocalized positively charged guanidinium functionality of this residue provides steering of the sugar substrate ManNAc for suitable placement in the active site and thus reaction with PEP.

The substrate capture mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase provides a mode for inhibition.

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 µM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.