The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans

Investor logo
Investor logo
Investor logo

Warning

This publication doesn't include Faculty of Education. It includes Faculty of Science. Official publication website can be found on muni.cz.
Authors

SEDLÁČEK Vojtěch KLUMPLER Tomáš MAREK Jaromír KUČERA Igor

Year of publication 2014
Type Article in Periodical
Magazine / Source PLOS ONE
MU Faculty or unit

Faculty of Science

Citation
Web http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0096262
Doi http://dx.doi.org/10.1371/journal.pone.0096262
Field Biochemistry
Keywords enzyme; flavin; crystal structure; mechanism
Description 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.
Related projects:

You are running an old browser version. We recommend updating your browser to its latest version.