Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 18.104.22.168). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77mM for CTP and 1.82mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.
- N-acetylneuraminic acid
- CMP-Neu5Ac synthetase
- sialic acids
1 This paper is dedicated to Professor J.A. Cabezas on the occasion of his retirement.
Abbreviations used: ACT, acylneuraminate cytidylyltransferase; BHIB, Brain Heart Infusion Broth; CMP-NeuAc, CMP-N-acetylneuraminic acid; CMP-NeuGc, CMP-N-glycolylneuraminic acid; Nbs2 (‘DTNB’), 5,5′-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; ManNAc, N-acetylmannosamine; −6-P, 6-phosphate; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; PEP, phosphoenolpyruvate; PHMB, p-hydroxymercuribenzoic acid; Pyr, pyruvate; UDP-GlcNAc, UDP-N-acetylglucosamine.
- The Biochemical Society, London ©2001