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

Wayne K Chou,Scott Dick,Warren W Wakarchuk,Martin E Tanner

doi:10.1074/jbc.m507483200

Wayne K Chou, Scott Dick + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m507483200

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Abstract

Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid). The presence of pseudaminic acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a pseudaminic acid synthase; the enzyme directly responsible for the biosynthesis of pseudaminic acid. Pseudaminic acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc), to form pseudaminic acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that pseudaminic acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for k(cat) of 0.65 +/- 0.01 s(-1), K(m)PEP of 6.5 +/- 0.4 microM, and K(m)6-deoxy-AltdiNAc of 9.5 +/- 0.7 microM. A mechanistic study on pseudaminic acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic acid synthase.

Highlights

Pseudaminic acid shares great structural similarity with another 9-carbon 2-keto-3-deoxy acid, N-acetylneuraminic acid (NeuNAc or sialic acid) (Fig. 1B)
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. 2a)
In this study we report the first identification and characterization of the activity of NeuB3 as a pseudaminic acid synthase that catalyzes the condensation of PEP with 6-deoxy-AltdiNAc to form pseudaminic acid

Summary

EXPERIMENTAL PROCEDURES

Materials and General Methods—Purine nucleoside phosphorylase and phosphoenolpyruvate were purchased from Sigma. 18O-Enriched water (95%) was purchased from Icon Isotopes. 2-Amino-6-mercapto7-methylpurine ribonucleoside was purchased from Berry and Associates. A solution of Tris-DCl buffer prepared in D2O (700 ␮l, 10 mM, pD 7.4) containing 6-deoxy-AltdiNAc (10 mM) and PEP (20 mM) was placed in an NMR tube. A solution of Tris-DCl buffer prepared in D2O (700 ␮l, 10 mM, pD 7.4) containing 6-deoxy-AltdiNAc (10 mM) and 20 mM [2-18O]PEP was placed in an NMR tube, and Chelex-100 resin (ϳ20 mg previously washed with D2O) was added. The solution was decanted from the Chelex resin and mixed with 50 mg of NeuB3 (buffer exchanged with the deuterated Tris-HCl buffer (pD 7.4)) and 1 mM. Pseudaminic acid was characterized using 1H, 13C, two-dimensional COSY, HMQC, and one-dimensional TOCSY NMR spectroscopy (in 10 mM deuterated phosphate buffer pD 7.4), and negative ESI-MS mass spectrometry. Controls included adding 10 mM EDTA or no additives (enzyme as isolated) to the kinetic assay

RESULTS AND DISCUSSION

Coupling constants ppm

Carbon signal ppm

Enzyme as isolated