Structure and characterisation of CMP-Kdn synthetase from the haptophyte microalgae <i>Prymnesium parvum</i>.

Abstract

Sialic acids - 9-carbon ulosonic acids - are implicated in many cell-cell and host-pathogen interactions due to their prevalent location at the non-reducing end of glycoconjugates. Sialic acids have recently been observed in microalgae, including the toxic bloom-forming <i>Prymnesium parvum</i>, which produces the deaminated sialic acid, ketodeoxynonulosonic acid (Kdn), through <i>de novo</i> biosynthesis. Here we report on the key CMP-sialic acid synthetase enzyme (CMAS), PpNeuA, which activates Kdn to its sugar nucleotide congener, CMP-Kdn. In the present study, the X-ray crystal structure of PpNeuA was determined to 1.8 Å resolution and shows that it adopts a similar overall fold to that of other sialic acid synthetase enzymes, with which it shares ca 30% amino acid sequence identity. PpNeuA specificity for Kdn is dependent upon Arg196, a hydrophilic residue that is only found in Kdn-specific sialic acid synthetases. R196L mutation switches the substrate preference of PpNeuA from Kdn to <i>N</i>-acetylneuraminic acid (Neu5Ac). Kinetic analysis shows that Arg196 plays both a role in substrate binding (impact on <i>K</i> _M) and catalysis (impact on <i>k</i> _cat). In the context of generating metabolic probes to identify the location and context (glycolipid vs glycoprotein) of Kdn in <i>P. parvum</i>, we also report on the ability of PpNeuA to accept both 5Az-Kdn and 9Az-Kdn as substrates.