3u7r
FerB - flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from Paracoccus denitrificansFerB - flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from Paracoccus denitrificans
Structural highlights
FunctionPublication Abstract from PubMedFerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases. The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.,Sedlacek V, Klumpler T, Marek J, Kucera I PLoS One. 2014 May 9;9(5):e96262. doi: 10.1371/journal.pone.0096262. eCollection , 2014. PMID:24817153[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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