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Literature summary for 1.6.5.2 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
Search for more literature for 1.6.5.2

Cloned(Commentary)

Cloned (Comment) Organism
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)

Crystallization (Comment) Organism
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

Protein Variants Comment Organism
E77A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
E77K site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
E77L site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
E77M site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
G115F site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
G115I site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
G118F site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
G118I site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
N18A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
N79A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
R13A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
R80E site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
R80K site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
R80L site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
R80M site-directed mutagenesis, almost inactive mutant Paracoccus denitrificans
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
R95A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Paracoccus denitrificans
R95E site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
S113A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Paracoccus denitrificans
S11A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
S16A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
Y46A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans
Y78A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Paracoccus denitrificans
Y78A site-directed mutagenesis, the mutation does not at all affect the enzyme activity Paracoccus denitrificans

KM Value [mM]

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

Localization

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 ?
-
 ?
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 ?
-
 ?
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
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

Organism UniProt Comment Textmining
Paracoccus denitrificans A1B9E3
-
-
Paracoccus denitrificans Pd 1222 A1B9E3
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 ?
-
 ?
additional information low activity with riboflavin instead of FMN Paracoccus denitrificans ?
-
 ?
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 ?
-
 ?
additional information low activity with riboflavin instead of FMN Paracoccus denitrificans Pd 1222 ?
-
 ?
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
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
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
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
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
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
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

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
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
additional information the enzyme is not NADPH-dependent, but NADH-dependent Paracoccus denitrificans
NAD+
-
 Paracoccus denitrificans
NADH
-
 Paracoccus denitrificans
riboflavin low activity with riboflavin instead of FMN Paracoccus denitrificans

General Information

General Information Comment Organism
evolution enzyme FerB is listed in the NADPH-dependent FMN reductase family, PF03358 Paracoccus denitrificans
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
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]

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