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The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.
- Source :
-
PloS one [PLoS One] 2014 May 09; Vol. 9 (5), pp. e96262. Date of Electronic Publication: 2014 May 09 (Print Publication: 2014). - Publication Year :
- 2014
-
Abstract
- FerB 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.
- Subjects :
- Amino Acid Sequence
Amino Acids chemistry
Amino Acids genetics
Amino Acids metabolism
Bacterial Proteins genetics
Bacterial Proteins metabolism
Binding Sites genetics
Biocatalysis
Catalytic Domain genetics
Crystallography, X-Ray
Flavin Mononucleotide chemistry
Flavin Mononucleotide metabolism
Flavins chemistry
Flavins metabolism
Flavoproteins chemistry
Flavoproteins genetics
Flavoproteins metabolism
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
NADH, NADPH Oxidoreductases classification
NADH, NADPH Oxidoreductases metabolism
NADP chemistry
NADP metabolism
Oxidation-Reduction
Paracoccus denitrificans genetics
Protein Binding
Protein Multimerization
Scattering, Small Angle
Sequence Homology, Amino Acid
X-Ray Diffraction
Bacterial Proteins chemistry
NADH, NADPH Oxidoreductases chemistry
Paracoccus denitrificans enzymology
Protein Structure, Tertiary
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 9
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 24817153
- Full Text :
- https://doi.org/10.1371/journal.pone.0096262