Discovery of novel 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase from methanarcheon Methanocaldococcus jannaschii.
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Cho, Myung Kook.
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This study explores the potential assimilatory sulfate reduction pathway in methanarcheon by investigating the gene product of the open reading frame Mj0066. We expressed and purified the gene product of Mj0066 of Methanocaldococcus jannaschii to explore its substrate specificity and the reaction kinetics. Kinetic studies revealed the wild-type enzyme specifically reduced PAPS with E. coli thioredoxin supplied as the electron donor. The Kᵐ, V_max, and k_cat/Kᵐ value was 15.9 μM, 0.09429 μMmg⁻¹min⁻¹, and 5571 M⁻¹1s⁻¹, respectively, at pH 8.0 and 30°C. Therefore, the gene product of Mj0066 was identified as a novel 5’-phosphoadenosine-phosphosulfate reductase. Furthermore, site directed mutagenesis of individual cysteines and redox titration were performed in order to investigate the catalytic mechanism. The combined studies show Cys337 plays a role in substrate binding while Cys19 and Cys22 are involved in electron transfer. Taken together, PAPS-reductase activity advocates the presence of a PAPS-utilizing assimilatory sulfate reduction pathway M. jannaschii.