Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni

Abstract

Escherichia coli NeuNAc (N-acetylneuraminic acid) synthase catalyses the condensation of PEP (phosphoenolpyruvate) and ManNAc (N-acetylmannosamine) to form NeuNAc and is encoded by the neuB gene. Campylobacter jejuni has three neuB genes, one of which is very similar to the E. coli neuB gene. We have characterized the C. jejuni neuraminic acid synthase with respect to acylamino sugar specificity and stereochemistry of the PEP condensation. We determined the specificity of C. jejuni NeuNAc synthase for N-acetylmannosamine, N-butanoylmannosamine, N-propionoylmannosamine and N-pentanoylmannosamine. We find that, although this enzyme exhibits similar K(m) values for N-acylmannosamine molecules with different N-acyl groups, the kcat/K(m) values decreased with increasing chain length. NeuNAc synthase is a member of a PEP-utilizing family of enzymes that form oxo acids from PEP and a monosaccharide. This family includes KDO 8-P (2-keto-3-deoxy-D-manno-octulosonate 8-phosphate) synthase and DAH 7-P (2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate) synthase. Both enzymes catalyse the condensation of the re face of the aldehyde group of the monosaccharide with the si face of the PEP molecule. The C. jejuni NeuNAc synthase catalysed the condensation of Z- and E-[3-2H]PEP with ManNAc, yielding (3S)-3-deutero-NeuNAc and (3R)-3-deutero-NeuNAc respectively. The condensation of Z-[3-F]PEP and ManNAc yielded (3S)-3-fluoro-NeuNAc. Results of our studies suggest that the C. jejuni NeuNAc synthase, similar to KDO 8-P synthase and DAH 7-P synthase, catalyses the condensation of the si face of PEP with the aldehyde sugar. The present study is the first stereochemical analysis of the reaction catalysed by a bacterial NeuNAc synthase.

Identification and Characterization of NeuB3 from Campylobacter jejuni as a Pseudaminic Acid Synthase

“…The extent of 18 O incorporation at the C-2 position was determined to be 54% by mass spectrometry: 16 O, 85). 31 P NMR spectroscopy was used to determine that the position of the label was at the C-2 position:…”

“…1A). Three genes that show homology to neuB were identified in C. jejuni, where one encoded a sialic acid synthase (neuB1) and the other two genes were thought to encode proteins that modify the flagellin proteins (neuB2 and neuB3) (8,16). NeuB3 shows 35.0% identity to the NeuB from Neisseria meningitidis and was found to be essential for the formation of flagella because an insertional mutation in the gene resulted in aflagellate C. jejuni (8).…”

“…Sialic acid synthase catalyzes the condensation of phosphoenolpyruvate with N-acetyl-D-mannosamine (ManNAc) to form sialic acid and phosphate (12)(13)(14). Recent studies have reported on both the structure of NeuB and its mechanism of action (15,16). Catalysis was demonstrated to proceed through an overall C-O bond cleavage process where the si-face of phosphoenolpyruvate (PEP) 2 initially attacks the aldehyde of the open chain form of ManNAc and water adds to the C-2 position forming a tetrahedral intermediate (Fig.…”

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.…”