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
elemental sulfur (S(0)) 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 S(0) 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 S(0) 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 S(0). 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 S(0)-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 S(0), suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S(0) was absent.
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Phurt
P
Harnvoravongchai
Hiroki
H
Kobori
Izumi
I
Orita
Satoshi
S
Nakamura
Tadayuki
T
Imanaka
Toshiaki
T
Fukui
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D013447:Sulfites / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D013447:Sulfites; D009247:NADH, NADPH Oxidoreductases; C042345:hydrogen sulfite