Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S⁰-Based Respiration and a Sulfur-Dependent Antioxidant System in Pyrococcus

Abstract

FAD and NAD(P)H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur-reducing anaerobic hyperthermophiles Pyrococcus horikoshii and Pyrococcus furiosus; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from P. horikoshii to 2.7 Å resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds. These results indicate that the likely in vivo substrates are NAD(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S(8)) in situ is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O(2) and/or H(2)O(2). As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S(0) respiration in Pyrococcus or Thermococcus but appears to participate in oxidative defense in the presence of S(0). When compared to the homologous Npsr enzyme from Shewanella loihica PV-4 and homologous enzymes known to reduce CoA disulfide, the phCoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates.

Elemental sulfur reduction in the deep‐sea vent thermophile, Thermovibrio ammonificans

“…The Cys43 residue is conserved in both enzymes and in the T. ammonificans NSR-like protein. Interestingly, the CoA persulfide/polysulfide reductase from the archaeon, Pyrococcus horikoshii (Herwald et al, 2013), shares close structural similarity with the NSR-like enzyme from T. ammonificans, contains a cysteine residue at position 43, and was shown to be capable of NAD(P)H-dependent polysulfide reduction (Fig. 5).…”

A broader active site in Pyrococcus horikoshii CoA disulfide reductase accommodates larger substrates and reveals evidence of subunit asymmetry

“…To further test the plasticity of the substrate channel, assays were run using glutathione disulfide as the substrate. Neither enzyme exhibited activity with GSSG .…”

“…The final product of the quad mutant titration is similar to the CoADR from S. aureus in which titration with excess NADPH yields reduction of only one monomer per dimer , although in S. aureus the FAD is reduced without the lag we saw in the quad mutant ph CoADR. S. aureus has a more open active site , unlike the wild‐type ph CoADR but similar to the quadruple mutant.…”

“…Future work with this NADH oxidase and the ph CoADR will explore the dimer interface in order to understand the communication between dimers that creates the asymmetry. The crystal structure of ph CoADR shows three residues from the other subunit stabilize the CoA substrate in the active site . In the E. faecalis NADH peroxidase, another member of the PNDOR family, the crystal structure reveals residues from the opposite subunit are integral in catalysis.…”

“…Wild‐type P. horikoshii CoADR ( ph CoADR) was expressed in BL21(DE3) Escherichia coli cells containing a pET24d plasmid with the gene and purified as previously described . A codon‐optimized gene for the quadruple mutant Y65A, Y66A, P67G, H367G (‘quad mutant’) was purchased from GenScript (Piscataway, NJ, USA) and inserted into a pET21b+ vector at the Nde I and Xho I restriction sites to create a final construct with a C‐terminal His tag.…”