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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