3nt6
Structure of the Shewanella loihica PV-4 NADH-dependent persulfide reductase C43S/C531S Double MutantStructure of the Shewanella loihica PV-4 NADH-dependent persulfide reductase C43S/C531S Double Mutant
Structural highlights
FunctionPublication Abstract from PubMedThe NADH-dependent persulfide reductase (Npsr), a recently discovered member of the PNDOR family of flavoproteins that contains a rhodanese domain in addition to the canonical flavoprotein reductase domain, is proposed to be involved in the dissimilatory reduction of S<sup>0</sup> in <i>Shewanella loihica</i> PV-4, a facultative anaerobe capable of utilizing a wide range of respiratory substrates. We have previously shown that polysulfide is able to act as a substrate for this enzyme, and a recently determined structure of a closely related enzyme (CoADR-Rhod from <i>Bacillus anthracis</i>) suggested the importance of a bound coenzyme A in the mechanism. The work described here shows that small thiol persulfides, such as CoA and glutathione persulfides, are the likely in vivo oxidizing substrates for Npsr is and suggests that persulfides may play a central role in the respiration of S0. C43S, C531S and C43,531S mutants were created to determine the role of the flavoprotein domain cysteine (C43) and the rhodanese domain cysteine (C531) in the mechanism of the enzyme. The absolute requirement for C43 for persulfide or DTNB reductase activity indicates that this residue is involved in S-S bond breakage. C531 contributes to, but is not required for, catalysis of DTNB reduction, while it is absolutely required for reduction of any persulfide substrates. Titrations of the enzyme with NADH, dithionite, titanium(III) and TCEP demonstrate the presence of a mixed-disulfide between C43 and a tightly bound CoA, and structures of the C43 and C43,531S mutants confirm that this coenzyme A remains tightly bound to the enzyme in the absence of a C43-CoA S-S bond. The 2.0 A structures of wild-type and mutant Npsr suggests a likely site for persulfide substrate binding and reaction with the rhodanese domain cysteine. Based on kinetic, titration and structural data a mechanism for the reduction of persulfides by Npsr is proposed. Characterization of an NADH-dependent persulfide reductase from <i>Shewanella loihica</i> PV-4: Implications for the mechanism of sulfur respiration via FAD-dependent enzymes.,Warner MD, Lukose V, Lee KH, Lopez K, Sazinsky M, Crane Iii EJ Biochemistry. 2010 Nov 23. PMID:21090815[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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