Structural and Mechanistic Analysis of Sialic Acid Synthase NeuB from Neisseria meningitidis in Complex with Mn2+, Phosphoenolpyruvate, and N-Acetylmannosaminitol

Abstract

In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an 18 O-labeling study that demonstrates the synthase operates via a COO bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (␤/␣) 8 barrel (TIM barrel)-type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 Å resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the Nacetylmannosamine carbonyl to the addition of PEP.

Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict

“…The conflict can be explained by the structure of the SAS holoenzyme. SAS monomer structure resembles an asymmetric dumbbell (16), and the active enzyme consists of a dimer or tetramer through juxtaposition of the N-terminal domain of one monomer with the Cterminal domain of another, forming the site for substrate (sugar) binding (28) between the interacting surfaces of the swapped domains of each monomer (Fig. S7).…”

“…Among these, type III AFPs (AFPIII) of various polar zoarcoid fishes (eelpouts, ocean pouts, and wolffishes) are homologous with the small C-terminal domain of sialic acid synthase (SAS) (14,15). SAS is an old cytoplasmic enzyme present in microbes (16) through vertebrates (17) that catalyzes intracellular synthesis of sialic acids from N-acetylmannosamine or Man-NAc-6-phosphate and phosphoenolpyruvate (16). In contrast, AFPIIIs are secreted plasma proteins that bind to invading ice crystals and arrest ice growth to prevent fish freezing (13).…”

PseG of Pseudaminic Acid Biosynthesis

“…Mechanistic studies have shown that the reaction proceeds via an anti-elimination of UDP to give the enzyme-bound intermediate 2-acetamidoglucal, followed by the syn-addition of water to give ManNAc (17,19). In a subsequent step the ManNAc is condensed with phosphoenolpyruvate by sialic acid synthase, NeuB, which is homologous to pseudaminic acid synthase and employs a similar reaction mechanism (20,21). Finally, the sialic acid is activated as its CMP form by CMP-sialic acid synthetase, NeuA (22,23).…”

“…Since the overall strategy employed in pseudaminic acid biosynthesis (the use of UDP-GlcNAc in ultimately forming a CMP-linked ␣-keto acid) is known to be the same as in sialic acid biosynthesis (24), and since the key synthases are closely related (13,20,21), it seemed possible that the hydrolases may also be homologous. Such an enzyme could catalyze the elimination of UDP from UDP-6-deoxy-AltdiNAc to give a glycal intermediate and then hydrate the double bond of the glycal with overall retention of stereochemistry at C-2 to give the hydrolysis product, 6-deoxy-AltdiNAc.…”

Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-Dgalacto- nonulosonic acid 9-phosphate synthase activities

“…Two distinct domains comprise the neuB gene products. The catalytic domain (TIM barrel domain) positioned N-terminally consists of ~270 AA and folds as a typical triose-phosphate isomerase (beta/alpha) 8 barrel (6). A linker of 11 AA connects it to a smaller, ~75 AA antifreeze-like domain (AFL domain), the name indicating its similarity to type III antifreeze proteins (7).…”