Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S⁰) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H₂S from S⁰ and free CoA-SH was observed, and the addition of enzyme and NADPH enhanced H₂S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA-SH acted to solubilize S⁰ by forming CoA persulfides, followed by reduction of an enzyme-S-S-CoA intermediate produced after both enzymatic and non-enzymatic evolution of H₂S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA-SH, implying a direct reaction with S⁰. TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA-SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S⁰-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S⁰, suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S⁰ was absent.