PURIFICATION, CLONING, AND EXPRESSION OF A CYTIDINE 5'-MONOPHOSPHATE N-ACETYLNEURAMINIC ACID SYNTHETASE FROM HAEMOPHILUS-DUCREYI

An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu Ac synthetase) was isolated from a Haemophilus ducreyi strain 35000 ce ll lysate and partially characterized. The enzyme catalyzes the reacti on of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar d onor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sia lic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the r ole of sialic acid in a. ducreyi pathogenesis, the gene encoding the C MP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synth etase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases, Th e gene was cloned into a T7 expression vector, the protein expressed i n Escherichia coli, and purified to apparent homogeneity by anion exch ange, Green 19 dye, and hydrophobic interaction chromatography. The fi nal step yielded 20 mg of pure protein/liter of culture, The protein h as a predicted molecular mass of 25440.6 Pa, which was confirmed by el ectrospray mass spectrometry (M(expt) = 25439.9 +/- 1.4 Pa). The enzym e appears to exist as a dimer by size exclusion chromatography. In con trast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also usi ng N-glycolylneuraminic acid as a substrate.