title: 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
abstract: Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.
authors.affiliation: Departments for Structural and Medicinal Biochemistry, University of Duisburg-Essen, 45117 Essen, Germany; MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.
authors.forename: Johannes
authors.initials: J
authors.lastname: van den Boom
authors.affiliation: Departments for Bioinformatics, ZMB, University of Duisburg-Essen, 45117 Essen, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Heider
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Martin
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pastore
authors.affiliation: Departments for Structural and Medicinal Biochemistry, University of Duisburg-Essen, 45117 Essen, Germany; MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom. Electronic address: jonathan.mueller@uni-due.de.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Mueller
mesh_terms: D000250:Adenosine Phosphosulfate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D013430:Sulfate Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D009097:Multienzyme Complexes; D000250:Adenosine Phosphosulfate; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase