Extended Data Fig. 8: In vitro reconstitution of the \({\mathbf{SO}}_{\mathbf{4}}^{{\mathbf{2-}}}\) reduction pathway to measure MtPAPSR activity.
From: Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus
a, Scheme of the coupled enzyme assay used to measure MtPAPSR activity via the oxidation of reduced methyl viologen (MVred) at 600 nm under an N2-atmosphere and at 45 °C. The enzymes were recombinantly expressed in E.coli, except for the pyrophosphatase, which was commercially obtained. The reaction mix contained all enzymes, metals (Mn2+, Mg2+), HEPES buffer at pH 7.0 and the substrates \({{\rm{SO}}}_{4}^{2-}\) and ATP. The reaction was started by the addition of \({{\rm{SO}}}_{4}^{2-}\). b, c, ‘Blank’ corresponds to a solution containing the buffer, the substrates, MVred and dithionite but no enzymes. ‘All components’ contained every compound shown in scheme (a). b, Substrate specificity of MtPAPSR. ‘No MtAPSK’ corresponds to all components except MtAPSK, which prevented the generation of PAPS from APS. With APS as the substrate, a specific enzymatic activity of 0.007 ± 0.001 µmol of oxidized MV.min−1.mg−1 of PAPSR was measured, which is only 6.54 % of the activity with the APSK (corresponds to Fig. 4a, +APS -ATPS -APSK). The difference can be attributed to an instability of the added APS. c, Impact of different concentrations of MtPAPSR on the activity: A fivefold addition of MtPAPSR resulted in a stimulation of the MV oxidation rate by 220 % (shown in dark green squares, ‘5 × more MtPAPSR’). After 30 minutes the five-fold addition of the MtPAPS−reductase led to aggregation, which is why the OD600nm started to increase after this time point. All experiments were performed in triplicates and represented as data mean ± s.d.
