Discovery of a novel adenosine 5'-phosphosulfate (APS) reductase from the methanarcheon Methanocaldococcus jannaschii.
Lee, Jong-Sun, 1979-
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This thesis presents the first discovery of adenosine 5'-phosphosulfate reductase (APR), a key enzyme of the sulfate reduction pathway, in the methanarchaeon Methanocaldococcus jannaschii. While the sulfate reduction pathway is present in other organisms, it is not expected to exist in methanarchaea because their habitats often already possess an abundance of reduced sulfur, particularly H₂S. However, the gene product of open reading frame (ORF) Mj0973 in M. jannaschii possesses sequence similarities with known APRs and 3'-phosphoadenosine-5'-phosphosulfate reductases (PAPRs) from various organisms. In order to further investigate this ORF, the gene Mj0973 from M. jannaschii was expressed and the resulting protein was purified. Kinetic studies revealed that the purified protein is able to reduce APS with E. coli thioredoxin (Trx) supplied as the electron donor, but is unable to reduce PAPS. The apparent Km, Vmax, and kcat/Km values at pH 8.0 and 30°C were 0.29 µM, 0.079µMmg⁻¹min⁻¹, and 299,655 M⁻¹s⁻¹, respectively. This observation of APR activity strongly indicates the presence of an APS-utilizing sulfate reduction pathway in the methanarchaeon M. jannaschii.