PAPS synthases - naturally fragile enzymes specifically stabilised by nucleotide binding

The activated sulphate donor 3'-phospho-adenosine-5'-phosphosulphate (PAPS) is needed for all biological sulphation reactions in eukaryotes with implications for the built-up of extracellular matrices, retroviral infection, protein modification and steroid metabolism. In multicellular organisms, PAPS is produced by bi-functional PAPS synthases where two enzymatic activities, an ATP sulphurylase and an APS kinase, reside on one polypeptide chain. A major question in the field is why the two human protein isoforms, PAPSS1 and -S2 cannot complement for each other. We have indications that these two proteins differ greatly in their protein stability as observed by differential fluorescence spectrometry (Thermofluor), unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism (CD). In presence of the nucleotide adenosine-5’-phosphosulphate (APS) one part of the protein is specifically stabilised in CD melting curves. Invertebrates only possess one PAPS synthase enzyme; hence, we asked whether the more stable or unstable behaviour is conserved in evolution. Therefore, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermally induced unfolding. By and large, PPS-1 behaved like the unstable human PAPSS2 protein with an apparent T50 below 40 °C. The protein stability seems to be a major contributing factor for PAPS availability that has not been anticipated so far.