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.

Cytidine 5¢-triphosphate synthase catalyses the ATPdependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH 3 -dependent CTP formation. For these mutants, the values of k cat /K m and k cat for glutamine-dependent CTP formation were reduced % 20-fold and % 10-fold, respectively, relative to wild-type enzyme; however, the value of K m for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis.CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or NH 3 as the nitrogen source (Scheme 1) [1,2]. This glutamine amidotransferase is a single polypeptide chain containing 545 amino acids and consisting of two domains. The C-terminal glutamine amide transfer (GAT) domain catalyses the hydrolysis of glutamine, and the nascent NH 3 derived from glutamine hydrolysis is transferred to the N-terminal synthase domain where the amination of UTP is catalysed [3,4]. CTPS belongs to the Triad family of glutamine amidotransferases [5,6] which utilizes a Cys-His-Glu triad to catalyse glutamine hydrolysis and also includes anthranilate synthase, carbamoyl phosphate synthase, formylglycinamidine synthase, GMP synthase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthase.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospholipids [8], CTPS is a recog...

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