Introduction The activated sulphate donor 3'-phospho- adenosine-5'-phosphosulphate (PAPS) is needed for all biological sulphation reactions in eukaryotes. In multicellular organisms, PAPS is produced by the bi-functional PAPS synthases that have come into the focus of biomedical research recently. We have indications that the only two human isoforms of this enzyme differ greatly in their protein stability; nucleotides seem to stabilise the proteins notably. Johannes van den Boom [email protected] Strukturelle und Medizinische Biochemie - ZMB PAPS synthases - naturally fragile enzymes specifically stabilised by nucleotide binding Figure 1: Schematic of the two-step PAPS production catalysed by PAPS synthases Figure 2: Chemical unfolding monitored by intrinsic tryptophan fluorescence. Moreover, inactivation kinetics confirmed a less stable PAPS synthase 2. Figure 5: Machine learning classifies invertebrate PAPS synthases as P2-type. In agreement, the C. elegans enzyme PPS-1 shows very similar behaviour to human P2. Figure 4: PAPS synthases are specifically stabilised by nucleotide binding Figure 6: The position-specific drop-in-performance values from the Random Forest were mapped onto the PAPSS1 crystal structure 1X6V. From these positions, several point mutants were created. The double swapping mutants Met70Phe60 + Thr85Ser75 resulted in a near complete reversal of the stability of the respective kinase domains. Figure 7: The respective residues are completely conserved within P1-/P2-type families. Invertebrate enzymes have similar, but not identical residues at these positions. Acknowledgement We cordially thank Peter Bayer for generous support. Steve Martin is acknowledged for professional help with spectroscopic measurements. Affiliations Structural and Medicinal Biochemistry and Bioinformatics, Centre for Medical Biotechnology (ZMB), Faculty of Biology, University of Duisburg-Essen, 45117 Essen, GERMANY Figure 3: CD spectra of human PAPS synthases. Figure 8: APS binding not only stabilises the PAPS synthase protein, but also efficiently suppresses its aggregation.
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Introduction The activated sulphate donor 3'-phospho-adenosine-5'-phosphosulphate (PAPS) is needed for all biological sulphation reactions in eukaryotes. In multicellular organisms, PAPS is produced by the bi-functional PAPS synthases that have come into the focus of biomedical research recently. We have indications that the only two human isoforms of this enzyme differ greatly in their protein stability; nucleotides seem to stabilise the proteins notably.
Johannes van den Boom [email protected] Strukturelle und Medizinische Biochemie - ZMB
PAPS synthases - naturally fragile enzymes specifically stabilised by nucleotide binding
Figure 1: Schematic of the two-step PAPS production catalysed by PAPS synthases
Figure 2: Chemical unfolding monitored by intrinsic tryptophan fluorescence. Moreover, inactivation kinetics confirmed a less stable PAPS synthase 2.
Figure 5: Machine learning classifies invertebrate PAPS synthases as P2-type. In agreement, the C. elegans enzyme PPS-1 shows very similar behaviour to
human P2.
Figure 4: PAPS synthases are specifically stabilised by nucleotide binding
Figure 6: The position-specific drop-in-performance values from the Random Forest were mapped onto the PAPSS1 crystal structure 1X6V. From these positions, several point mutants were created. The double swapping mutants Met70Phe60 + Thr85Ser75 resulted in a near complete reversal of the stability of the respective kinase domains.
Figure 7: The respective residues are completely conserved within P1-/P2-type families. Invertebrate enzymes have similar, but not identical residues at these positions.
Acknowledgement We cordially thank Peter Bayer for generous support. Steve Martin is acknowledged for professional help with spectroscopic measurements.
Affiliations Structural and Medicinal Biochemistry and Bioinformatics, Centre for Medical Biotechnology (ZMB), Faculty of Biology, University of Duisburg-Essen, 45117 Essen, GERMANY
Figure 3: CD spectra of human PAPS synthases.
Figure 8: APS binding
not only stabilises the PAPS synthase
protein, but also efficiently
suppresses its aggregation.
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