4ocg | pdb_00004ocg
Structure of the Shewanella loihica PV-4 NADH-dependent persulfide reductase F161A Mutant
Structural highlights
Publication Abstract from PubMedThe NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4 is a member of the single cysteine-containing subset of the family of disulfide reductases represented by glutathione reductase. We have determined the kinetics of the reductive half-reaction of the enzyme with NADH using stopped-flow spectroscopy and kinetic isotope effects, and these results indicate that the reductive and oxidative half-reactions are both partially rate-limiting for enzyme turnover. During reaction with NADH, the reduced nucleotide appears to bind rapidly in an unproductive conformation, followed by the formation of a productive E.NADH complex and subsequent electron transfer to FAD. F161 of Npsr fills the space in which the nicotinamide ring of NADH would be expected to bind. We have shown that while this residue is not absolutely required for catalysis, it does assist in the forward commitment to catalysis through its role in the reductive half reaction, where it appears to enhance hydride transfer in the productive E.NADH complex. While the fluorescence and absorbance spectra of the stable redox forms of the wild-type and F161A mutant enzymes are similar, intermediates formed during reduction and turnover have different characteristics and appear to indicate that the enzyme-NADH complex formed just prior to hydride transfer on the F161A enzyme has weaker FAD-NADH interactions than the wild-type enzyme, consistent with a "looser" enzyme-NADH complex. The 2.7A crystal structure of the F161A mutant was determined, and shows that the nicotinamide ring of NADH would have the expected freedom of motion in the more open NADH binding cavity. Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: Formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes.,Lee KH, Humbarger S, Bahnvadia R, Sazinsky MH, Crane EJ 3rd Biochim Biophys Acta. 2014 Sep;1844(9):1708-17. doi:, 10.1016/j.bbapap.2014.06.013. Epub 2014 Jun 26. PMID:24981797[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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