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.

Scheme 1. Reaction catalysed by C. jejuni NeuNAc synthase

Figure 1. SDS/PAGE of purified C. jejuni NeuNAc synthase (Cjsas)

MM STD, molecular-mass standards (kDa).

Figure 2. ManNAc analogues

Figure 3. ¹H-NMR spectra of NeuNAc

¹H-NMR spectra of (a) NeuNAc, (b) (3R)-[3-²H]NeuNAc obtained from E-[3-²H]PEP, and (c) (3S)-[3-²H]NeuNAc obtained from Z-[3-²H]PEP. Only the regions exhibiting signals for C-3 protons are shown (the signal at 1.78 p.p.m. is from H_a and the signal at 2.19 p.p.m. is from H_eq). The signal at 2 p.p.m. is from the methyl group of N-acyl side chain.

Figure 4. ¹⁹F-NMR spectra obtained for the reaction mixture containing Z-[3-F]PEP, ManNAc, MnCl₂ and C. jejuni NeuNAc synthase

Signal at −140 p.p.m. is from Z-[3-F]PEP and the signal at −199.4 p.p.m. is from the product (3S)-[3-F]NeuNAc.

Figure 5. ¹⁹F-NMR spectra obtained for the reaction mixture containing E- and Z-[3-F]PEP (3:2 mixture), ManNAc, MnCl₂ and C. jejuni NeuNAc synthase

The signal at −140 p.p.m. is from Z-[3-F]PEP and the signal at −150 p.p.m. is from E-[3-F]PEP. The signal at −199.4 p.p.m. is from the product (3S)-[3-F]NeuNAc (formed from the Z-isomer).

Scheme 2. Condensation of PEP with ManNAc by NeuNAc synthase