Some of the enzymes involved in the biosynthesis of the Escherichia coli K1, a pathogen, polysaccharide is central to neuraminic acid metabolism in bacteria and mammals. The expression and function of CMP-neuAc synthetase and a protein of undefined function, P7 is being investigated. The role of the 2 CMP-neuAc synthetase has cysteine residues in function was investigated by chemical modification and site directed mutagenesis. Reducing agents and iodoactate have no effect on the rate of heat inactivation suggesting that a disulfide is not critical for stability. Enzyme can be inactivated by sulfhydryl specific reagents such PCMB and dithiodipyridine. Site directed mutagenesis of either cys-129 or cys-329 to glycine results in a marked decrease in activity, thus both sulfhydryls are important for full activity. Comparison of the nucleotide sequences of CMP-neuAc synthetase and the 2 CMP-KDO synthetase enzymes produced by encapsulated E. coli suggest a consensus sequence in the amino termini. Random mutagensis suggest that some the residues may be important for activity. Of 9 (out of 15 total) arginine residues mutated to glycine only the arg-12 contained in the amino terminal consensus affected activity. Experiments are underway to define the role of arginine-12 in catalysis. The CMP-neuAc synthetase of Neisseria meningitidis has been partially purified. The amino terminus of this enzyme will be sequenced and compared with the current consensus. Analysis of available nucleotide sequence data from our laboratory and the University of Rochester suggest that the genes encoding CMP-neuAc synthetase and P7 are part of an operon ending with P7. The promoter for this operon has not been located. Based on these data and previous experiments, CMP-neuAc and P7 may be translationally coupled. Fusion proteins of P7 and ?-galactosidase are being constructed to test this hypothesis and to aid in the purification of P7.
