| SEDLÁČEK, Vojtěch, Tomáš KLUMPLER, Jaromír MAREK a Igor KUČERA. The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans. PLOS ONE. Public Library of Science, 2014, roč. 9, č. 5, s. 1-13. ISSN 1932-6203. Dostupné z: https://dx.doi.org/10.1371/journal.pone.0096262. |
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@article{1185572,
author = {Sedláček, Vojtěch and Klumpler, Tomáš and Marek, Jaromír and Kučera, Igor},
article_number = {5},
doi = {http://dx.doi.org/10.1371/journal.pone.0096262},
keywords = {enzyme; flavin; crystal structure; mechanism},
language = {eng},
issn = {1932-6203},
journal = {PLOS ONE},
title = {The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans},
url = {http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0096262},
volume = {9},
year = {2014}
}
TY - JOUR
ID - 1185572
AU - Sedláček, Vojtěch - Klumpler, Tomáš - Marek, Jaromír - Kučera, Igor
PY - 2014
TI - The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans
JF - PLOS ONE
VL - 9
IS - 5
SP - 1-13
EP - 1-13
PB - Public Library of Science
SN - 19326203
KW - enzyme
KW - flavin
KW - crystal structure
KW - mechanism
UR - http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0096262
L2 - http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0096262
N2 - 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.
ER -
SEDLÁČEK, Vojtěch, Tomáš KLUMPLER, Jaromír MAREK a Igor KUČERA. The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans. \textit{PLOS ONE}. Public Library of Science, 2014, roč.~9, č.~5, s.~1-13. ISSN~1932-6203. Dostupné z: https://dx.doi.org/10.1371/journal.pone.0096262.
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