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 (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 | 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 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytoplasm | - |
Paracoccus denitrificans | 5737 | - |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Paracoccus denitrificans | A1B9E3 | - |
- |
Paracoccus denitrificans Pd 1222 | A1B9E3 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) | Paracoccus denitrificans |
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 |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
stopped-flow spectrophotometry at 10°C | Paracoccus denitrificans |
25 | - |
assay at | Paracoccus denitrificans |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Paracoccus denitrificans |
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 | 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 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 |