Literature summary for 1.3.1.6 extracted from

Rothery, E.L.; Mowat, C.G.; Miles, C.S.; Walkinshaw, M.D.; Reid, G.A.; Chapman, S.K.
Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina (2003), Biochemistry, 42, 13160-13169.

Cloned(Commentary)

Cloned (Comment)	Organism
gene fccA, subcloning in Escherichia coli, expression of wild-type and mutant flavocytochrome enzymes in the enzyme-deficient strain DELTAfccA EG301	Shewanella frigidimarina

Crystallization (Commentary)

Crystallization (Comment)	Organism
mutant enzyme H61A and H61M, hanging drop vapour diffusion method, protein solution: 6 mg/ml, 10 mM Tris-HCl, pH 8.5, well solution: 100 mM Tris-HCl, pH 7.4-8.5, 4°C, 80 mM NaCl, 16-19% PEG 8000, 10 mM fumarate, equal volume of 0.002 ml of protein solution and well solution, 10 days, cryoprotectant solution: 100 mM sodium acetate, pH 6.5, 20% PEG 8000, 10 mM fumarate, 80 mM NaCl, and 23% glycerol, X-ray diffraction structure determination and analysis at 2.1 A and 2.2 A resolution, respectively, molecular replacement	Shewanella frigidimarina

Crystallization (Comment)

Organism

mutant enzyme H61A and H61M, hanging drop vapour diffusion method, protein solution: 6 mg/ml, 10 mM Tris-HCl, pH 8.5, well solution: 100 mM Tris-HCl, pH 7.4-8.5, 4°C, 80 mM NaCl, 16-19% PEG 8000, 10 mM fumarate, equal volume of 0.002 ml of protein solution and well solution, 10 days, cryoprotectant solution: 100 mM sodium acetate, pH 6.5, 20% PEG 8000, 10 mM fumarate, 80 mM NaCl, and 23% glycerol, X-ray diffraction structure determination and analysis at 2.1 A and 2.2 A resolution, respectively, molecular replacement

Shewanella frigidimarina

Protein Variants

Protein Variants	Comment	Organism
H61A	site-directed mutagenesis, 5-10fold reduced kcat compared to the wild-type enzyme, elevated pH optimum, electrochemical analysis, crystal structure analysis, altered ligand binding of hemes and heme iron, 80% of wild-type activity can be recovered by addition of exogenous imidazole, overview	Shewanella frigidimarina
H61M	site-directed mutagenesis, 5-10fold reduced kcat compared to the wild-type enzyme, elevated pH optimum, electrochemical analysis, crystal structure analysis, altered ligand binding of hemes and heme iron, 80% of wild-type activity can be recovered by addition of exogenous imidazole, overview	Shewanella frigidimarina

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	pre-steady-state kinetics, solvent isotope effect, wild-type and mutant enzymes	Shewanella frigidimarina
0.005	-	fumarate	mutant H61M, pH 7.2 and pH 7.5, 25°C, and mutant H61A, pH 6.0, pH 7.2, pH 7.5, and pH 9.0, 25°C	Shewanella frigidimarina
0.007	-	fumarate	wild-type enzyme, pH 9.0, 25°C	Shewanella frigidimarina
0.009	-	fumarate	mutant H61A, pH 9.0, 25°C	Shewanella frigidimarina
0.01	-	fumarate	mutant H61M, pH 6.0, 25°C	Shewanella frigidimarina
0.025	-	fumarate	wild-type enzyme, pH 7.2, 25°C	Shewanella frigidimarina
0.028	-	fumarate	wild-type enzyme, pH 7.5, 25°C	Shewanella frigidimarina
0.043	-	fumarate	wild-type enzyme, pH 6.0, 25°C	Shewanella frigidimarina

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
soluble	-	Shewanella frigidimarina	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	enzyme contains 4 bis-His-ligated, c-type hemes, His61 is involved in binding, analysis of crystal structure	Shewanella frigidimarina

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
63033	-	x * 63033, wild-type enzyme, mass spectrometry	Shewanella frigidimarina

Organism

Organism	UniProt	Comment	Textmining
Shewanella frigidimarina	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant flavocytochrome enzymes from enzyme-deficient strain DELTAfccA EG301	Shewanella frigidimarina

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
fumarate + NADH + H+	electron transfer mechanism	Shewanella frigidimarina	succinate + NAD+	-	?

Subunits

Subunits	Comment	Organism
?	x * 63033, wild-type enzyme, mass spectrometry	Shewanella frigidimarina

Synonyms

Synonyms	Comment	Organism
fumarate reductase	-	Shewanella frigidimarina

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Shewanella frigidimarina

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
15	-	fumarate	mutant H61M, pH 9.0, 25°C	Shewanella frigidimarina
29	-	fumarate	mutant H61A, pH 9.0, 25°C	Shewanella frigidimarina
47	-	fumarate	mutant H61A, pH 7.5, 25°C	Shewanella frigidimarina
49	-	fumarate	mutant H61A, pH 7.2, 25°C	Shewanella frigidimarina
96	-	fumarate	mutant H61M, pH 7.5, 25°C	Shewanella frigidimarina
100	-	fumarate	mutant H61M, pH 6.0, 25°C	Shewanella frigidimarina
116	-	fumarate	mutant H61A, pH 6.0, 25°C	Shewanella frigidimarina
198	-	fumarate	mutant H61M, pH 7.2, 25°C	Shewanella frigidimarina
210	-	fumarate	wild-type enzyme, pH 9.0, 25°C	Shewanella frigidimarina
370	-	fumarate	wild-type enzyme, pH 7.5, 25°C	Shewanella frigidimarina
509	-	fumarate	wild-type enzyme and mutant H505Y, pH 7.2, 25°C	Shewanella frigidimarina
658	-	fumarate	wild-type enzyme and mutant H505A, pH 6.0, 25°C	Shewanella frigidimarina

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.2	-	assay at	Shewanella frigidimarina

Cofactor

Cofactor	Comment	Organism
FAD	binding structure, FAD content of wild-type and mutant enzymes, overview	Shewanella frigidimarina
heme	enzyme contains 4 bis-His-ligated, c-type hemes, His61 is involved in binding, analysis of crystal structure, fumarate dependent reoxidation of the heme groups in mutants H61A and H61M are affected	Shewanella frigidimarina
NADH	-	Shewanella frigidimarina