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Literature summary extracted from

  • Sedlacek, V.; Klumpler, T.; Marek, J.; Kucera, I.
    The structural and functional basis of catalysis mediated by NAD(P)H acceptor oxidoreductase (FerB) of Paracoccus denitrificans (2014), PLoS ONE, 9, e96262 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.6.5.2 gene ferB, sequence comparisons and phylogenetic tree, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Paracoccus denitrificans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.6.5.2 purified recombinant wild-type andd selenomethionine-labeled His6-tagged wild-type enzyme in complex with NADH, X-ray diffraction structure determination and analysis at 1.4-1.75 A resolution, multiple wavelength anomalous dispersion using selenium anomalous signal, modeling Paracoccus denitrificans

Protein Variants

EC Number Protein Variants Comment Organism
1.6.5.2 E77A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 E77K site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 E77L site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
1.6.5.2 E77M site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 G115F site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 G115I site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 G118F site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 G118I site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 N18A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 N79A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 R13A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 R80E site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 R80K site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 R80L site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
1.6.5.2 R80M site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
1.6.5.2 R95A site-directed mutagenesis, replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity of the mutant towards external flavins compared to the wild-type Paracoccus denitrificans
1.6.5.2 R95A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 R95E site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 S113A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 S11A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 S16A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 Y46A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
1.6.5.2 Y78A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Paracoccus denitrificans
1.6.5.2 Y78A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.5.2 additional information
-
 additional information steady-state kinetics, stopped-flow spectrophotometry Paracoccus denitrificans
1.6.5.2 0.02
-
 NADH pH 7.4, 25°C, recombinant mutant R95A Paracoccus denitrificans
1.6.5.2 0.022
-
 NADH pH 7.4, 25°C, recombinant wild-type enzyme Paracoccus denitrificans
1.6.5.2 0.03
-
 NADH pH 7.4, 25°C, recombinant mutant E77K Paracoccus denitrificans
1.6.5.2 0.03
-
 NADH pH 7.4, 25°C, recombinant mutant R80E Paracoccus denitrificans
1.6.5.2 0.038
-
 NADH pH 7.4, 25°C, recombinant mutant G118I Paracoccus denitrificans
1.6.5.2 0.04
-
 NADH pH 7.4, 25°C, recombinant mutant Y78A Paracoccus denitrificans
1.6.5.2 0.06
-
 NADH pH 7.4, 25°C, recombinant mutant S113A Paracoccus denitrificans
1.6.5.2 0.07
-
 NADH pH 7.4, 25°C, recombinant mutant R95E Paracoccus denitrificans
1.6.5.2 0.085
-
 NADH pH 7.4, 25°C, recombinant mutant G118F Paracoccus denitrificans
1.6.5.2 0.18
-
 NADH pH 7.4, 25°C, recombinant mutant E77A Paracoccus denitrificans
1.6.5.2 0.25
-
 NADH pH 7.4, 25°C, recombinant mutant E77L Paracoccus denitrificans
1.6.5.2 0.35
-
 NADH pH 7.4, 25°C, recombinant mutant R80K Paracoccus denitrificans
1.6.5.2 0.41
-
 NADH pH 7.4, 25°C, recombinant mutant G115F Paracoccus denitrificans
1.6.5.2 0.42
-
 NADH pH 7.4, 25°C, recombinant mutant E77M Paracoccus denitrificans
1.6.5.2 0.58
-
 NADH pH 7.4, 25°C, recombinant mutant R80L Paracoccus denitrificans
1.6.5.2 0.63
-
 NADH pH 7.4, 25°C, recombinant mutant G115I Paracoccus denitrificans
1.6.5.2 0.78
-
 NADH pH 7.4, 25°C, recombinant mutant R80M Paracoccus denitrificans

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.6.5.2 cytoplasm
-
 Paracoccus denitrificans 5737
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.6.5.2 additional information Paracoccus denitrificans the flavoprotein ferric reductase B, FerB, from Paracoccus denitrificans is one of two major enzymes able to reduce Fe(III)-ligand complexes when NADH is the electron donor. The protein is also active as a chromate reductase and, to a substantially greater extent, as a quinone reductase ?
-
 ?
1.6.5.2 additional information Paracoccus denitrificans Pd 1222 the flavoprotein ferric reductase B, FerB, from Paracoccus denitrificans is one of two major enzymes able to reduce Fe(III)-ligand complexes when NADH is the electron donor. The protein is also active as a chromate reductase and, to a substantially greater extent, as a quinone reductase ?
-
 ?
1.6.5.2 NADH + H+ + a quinone Paracoccus denitrificans hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring NAD+ + a hydroquinone
-
 r
1.6.5.2 NADH + H+ + a quinone Paracoccus denitrificans Pd 1222 hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring NAD+ + a hydroquinone
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.6.5.2 Paracoccus denitrificans A1B9E3
-
-
1.6.5.2 Paracoccus denitrificans Pd 1222 A1B9E3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.6.5.2 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Paracoccus denitrificans

Reaction

EC Number Reaction Comment Organism Reaction ID
1.6.5.2 NAD(P)H + H+ + a quinone = NAD(P)+ + a hydroquinone the activity obeys the standard bi-bi ping-pong reaction mechanism Paracoccus denitrificans
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.5.2 additional information the flavoprotein ferric reductase B, FerB, from Paracoccus denitrificans is one of two major enzymes able to reduce Fe(III)-ligand complexes when NADH is the electron donor. The protein is also active as a chromate reductase and, to a substantially greater extent, as a quinone reductase Paracoccus denitrificans ?
-
 ?
1.6.5.2 additional information low activity with riboflavin instead of FMN Paracoccus denitrificans ?
-
 ?
1.6.5.2 additional information the flavoprotein ferric reductase B, FerB, from Paracoccus denitrificans is one of two major enzymes able to reduce Fe(III)-ligand complexes when NADH is the electron donor. The protein is also active as a chromate reductase and, to a substantially greater extent, as a quinone reductase Paracoccus denitrificans Pd 1222 ?
-
 ?
1.6.5.2 additional information low activity with riboflavin instead of FMN Paracoccus denitrificans Pd 1222 ?
-
 ?
1.6.5.2 NADH + H+ + 2,3-dimethoxy-5-methyl-1,4-benzoquinone i.e. ubiquinone-10, UQ-10, a quinone electron acceptor Paracoccus denitrificans NAD+ + 2,3-dimethoxy-5-methyl-1,4-benzoquinol
-
 r
1.6.5.2 NADH + H+ + 2,3-dimethoxy-5-methyl-1,4-benzoquinone i.e. ubiquinone-10, UQ-10, a quinone electron acceptor Paracoccus denitrificans Pd 1222 NAD+ + 2,3-dimethoxy-5-methyl-1,4-benzoquinol
-
 r
1.6.5.2 NADH + H+ + 2,3-dimethoxy-5-methyl-2,5-cyclohexadiene-1,4-dione i.e. ubiquinone-0, UQ-0, a quinone electron acceptor Paracoccus denitrificans NAD+ + 2,3-dimethoxy-5-methylbenzene-1,4-diol
-
 r
1.6.5.2 NADH + H+ + a quinone hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring Paracoccus denitrificans NAD+ + a hydroquinone
-
 r
1.6.5.2 NADH + H+ + a quinone hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. The first, reductive half-reaction of flavin cofactor is rate-limiting Paracoccus denitrificans NAD+ + a hydroquinone
-
 r
1.6.5.2 NADH + H+ + a quinone hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring Paracoccus denitrificans Pd 1222 NAD+ + a hydroquinone
-
 r
1.6.5.2 NADH + H+ + a quinone hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. The first, reductive half-reaction of flavin cofactor is rate-limiting Paracoccus denitrificans Pd 1222 NAD+ + a hydroquinone
-
 r

Subunits

EC Number Subunits Comment Organism
1.6.5.2 dimer 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 Paracoccus denitrificans
1.6.5.2 More occurrence of a dimer-tetramer equilibrium Paracoccus denitrificans

Synonyms

EC Number Synonyms Comment Organism
1.6.5.2 FerB
-
 Paracoccus denitrificans
1.6.5.2 ferric reductase B
-
 Paracoccus denitrificans
1.6.5.2 NAD(P)H:acceptor oxidoreductase
-
 Paracoccus denitrificans
1.6.5.2 Pden_4071
-
 Paracoccus denitrificans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.6.5.2 additional information
-
 stopped-flow spectrophotometry at 10°C Paracoccus denitrificans
1.6.5.2 25
-
 assay at Paracoccus denitrificans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6.5.2 0.072
-
 NADH pH 7.4, 25°C, recombinant mutant E77L Paracoccus denitrificans
1.6.5.2 0.142
-
 NADH pH 7.4, 25°C, recombinant mutant R80L Paracoccus denitrificans
1.6.5.2 0.275
-
 NADH pH 7.4, 25°C, recombinant mutant R80E Paracoccus denitrificans
1.6.5.2 0.302
-
 NADH pH 7.4, 25°C, recombinant mutant R80M Paracoccus denitrificans
1.6.5.2 0.75
-
 NADH pH 7.4, 25°C, recombinant mutant E77K Paracoccus denitrificans
1.6.5.2 1.7
-
 NADH pH 7.4, 25°C, recombinant mutant G115I Paracoccus denitrificans
1.6.5.2 2.2
-
 NADH pH 7.4, 25°C, recombinant mutant G115F Paracoccus denitrificans
1.6.5.2 2.5
-
 NADH pH 7.4, 25°C, recombinant mutant E77M Paracoccus denitrificans
1.6.5.2 9.5
-
 NADH pH 7.4, 25°C, recombinant mutant R80K Paracoccus denitrificans
1.6.5.2 12
-
 NADH pH 7.4, 25°C, recombinant mutant G118I Paracoccus denitrificans
1.6.5.2 17.9
-
 NADH pH 7.4, 25°C, recombinant mutant E77A Paracoccus denitrificans
1.6.5.2 21.1
-
 NADH pH 7.4, 25°C, recombinant mutant R95E Paracoccus denitrificans
1.6.5.2 76.7
-
 NADH pH 7.4, 25°C, recombinant mutant S113A Paracoccus denitrificans
1.6.5.2 77
-
 NADH pH 7.4, 25°C, recombinant mutant G118F Paracoccus denitrificans
1.6.5.2 146
-
 NADH pH 7.4, 25°C, recombinant mutant R95A Paracoccus denitrificans
1.6.5.2 196
-
 NADH pH 7.4, 25°C, recombinant mutant Y78A Paracoccus denitrificans
1.6.5.2 258
-
 NADH pH 7.4, 25°C, recombinant wild-type enzyme Paracoccus denitrificans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.6.5.2 7.4
-
 assay at Paracoccus denitrificans

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.5.2 FMN flavin mononucleotide groups are non-covalently bound at the opposite sides of the subunit interface of the enzyme dimer, binding site structure analysis, overview. The flavin is required for activity, residue Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. interaction of apoenzyme with the FMN phosphate significantly stabilizes the complex with the enzyme. Hydride transfer occurs from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. The first, reductive half-reaction of flavin cofactor is rate-limiting. Importance of residue Ser11, Arg13, Ser16 and Asn18 for FMN binding, and of the tyrosine residues Tyr46 and Tyr78 and possibly also Ser113 for capturing the isoalloxazine ring of FMN Paracoccus denitrificans
1.6.5.2 additional information the enzyme is not NADPH-dependent, but NADH-dependent Paracoccus denitrificans
1.6.5.2 NAD+
-
 Paracoccus denitrificans
1.6.5.2 NADH
-
 Paracoccus denitrificans
1.6.5.2 riboflavin low activity with riboflavin instead of FMN Paracoccus denitrificans

General Information

EC Number General Information Comment Organism
1.6.5.2 evolution enzyme FerB is listed in the NADPH-dependent FMN reductase family, PF03358 Paracoccus denitrificans
1.6.5.2 additional information importance of residue Ser11, Arg13, Ser16 and Asn18 for FMN binding, and of the tyrosine residues Tyr46 and Tyr78 and possibly also Ser113 for capturing the isoalloxazine ring of FMN. FMN and NADH docking, overview Paracoccus denitrificans
1.6.5.2 physiological function 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 Paracoccus denitrificans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.6.5.2 0.24
-
 NADH pH 7.4, 25°C, recombinant mutant R80L Paracoccus denitrificans
1.6.5.2 0.29
-
 NADH pH 7.4, 25°C, recombinant mutant E77L Paracoccus denitrificans
1.6.5.2 0.39
-
 NADH pH 7.4, 25°C, recombinant mutant R80M Paracoccus denitrificans
1.6.5.2 2.7
-
 NADH pH 7.4, 25°C, recombinant mutant G115I Paracoccus denitrificans
1.6.5.2 5.4
-
 NADH pH 7.4, 25°C, recombinant mutant G115F Paracoccus denitrificans
1.6.5.2 6
-
 NADH pH 7.4, 25°C, recombinant mutant E77M Paracoccus denitrificans
1.6.5.2 9.2
-
 NADH pH 7.4, 25°C, recombinant mutant R80E Paracoccus denitrificans
1.6.5.2 25
-
 NADH pH 7.4, 25°C, recombinant mutant E77K Paracoccus denitrificans
1.6.5.2 99
-
 NADH pH 7.4, 25°C, recombinant mutant E77A Paracoccus denitrificans
1.6.5.2 302
-
 NADH pH 7.4, 25°C, recombinant mutant R95E Paracoccus denitrificans
1.6.5.2 316
-
 NADH pH 7.4, 25°C, recombinant mutant G118I Paracoccus denitrificans
1.6.5.2 906
-
 NADH pH 7.4, 25°C, recombinant mutant G118F Paracoccus denitrificans
1.6.5.2 1278
-
 NADH pH 7.4, 25°C, recombinant mutant S113A Paracoccus denitrificans
1.6.5.2 2767
-
 NADH pH 7.4, 25°C, recombinant mutant R80K Paracoccus denitrificans
1.6.5.2 4900
-
 NADH pH 7.4, 25°C, recombinant mutant Y78A Paracoccus denitrificans
1.6.5.2 7292
-
 NADH pH 7.4, 25°C, recombinant mutant R95A Paracoccus denitrificans
1.6.5.2 11727
-
 NADH pH 7.4, 25°C, recombinant wild-type enzyme Paracoccus denitrificans