BRENDA - Enzyme Database

Enhanced catalytic properties of novel (alphabetagamma)2 heterohexameric Rhodobacter capsulatus xanthine dehydrogenase by separate expression of the redox domains in Escherichia coli

Wang, C.; Li, G.; Zhang, C.; Xing, X.; Biochem. Eng. J. 119, 1-8 (2017)
No PubMed abstract available

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.17.1.4
genes xdhA-C, development of a method that translates active Rhodobacter capsulatus (alphabetagamma)2 XDH by directly expressing the iron-sulfur domain, the flavin adenine dinucleotide domain and the sulfurated molybdenum domain as three separate proteins in Escherichia coli, recombinant expression of wild-type enzyme and mutant enzymes, i.e. an (alphabetagamma)2 heterohexameric enzyme and two (alphabetagamma)2 XDH variants, in Escherichia coli
Rhodobacter capsulatus
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.17.1.4
additional information
two (alphabetagamma)2 XDH variants, Split166 and Split178, are designed and constructed by splitting the small subunit (alphabeta)2 XDH at the N- and C-terminal ends of the L167-A178 peptide linking the iron-sulfur clusters and flavin adenine dinucleotide domains, respectively. Subunit composition of recombinant wild-type and split XDHsAs, overview. As for the co-substrate NAD+, mutant Split178 has a 1.07fold increased catalytic efficiency, while Split166 has a 3.8fold decreased catalytic efficiency compared to the wild-type XDH, for the substrate xanthine, the Split178 variant shows 1.21fold increased turnover number and 1.66fold increased catalytic efficiency, while the mutant Split166 shows a 4.31fold decrease in comparison to the wild-type enzyme
Rhodobacter capsulatus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.17.1.4
additional information
-
additional information
Michaelis-Menten steady-state kinetics, overview
Rhodobacter capsulatus
1.17.1.4
0.036
-
NAD+
pH 8.5, 40°C, Split166 mutant; pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
0.044
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
0.055
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
0.068
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
0.096
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.17.1.4
Fe2+
in the [2Fe-2S] center
Rhodobacter capsulatus
1.17.1.4
Molybdenum
in the molybdenum cofactor
Rhodobacter capsulatus
1.17.1.4
additional information
the purified wild-type XDH contains 2.80 iron, 0.94 FAD, and 0.72 Moco per (alphabeta)2 tetrameric subunit, Split178 has 2.73 iron, 0.95 FAD, and 0.70 Moco per (alphabetagamma)2 hexameric subunit, while Split166 incorporates 3.51 iron, 0.95 FAD, and 0.95 Moco per (alphabetagamma)2 hexamer
Rhodobacter capsulatus
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.17.1.4
xanthine + NAD+ + H2O
Rhodobacter capsulatus
-
urate + NADH + H+
-
-
?
1.17.1.4
xanthine + NAD+ + H2O
Rhodobacter capsulatus CGMCC 1.3366
-
urate + NADH + H+
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.17.1.4
Rhodobacter capsulatus
O54050 AND O54051 AND Q9X7K2
small and large subunits encoded by genes xdhA and xdhB, and chaperone encoded by gene xdhC
-
1.17.1.4
Rhodobacter capsulatus CGMCC 1.3366
O54050 AND O54051 AND Q9X7K2
small and large subunits encoded by genes xdhA and xdhB, and chaperone encoded by gene xdhC
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.17.1.4
xanthine + NAD+ + H2O
-
744255
Rhodobacter capsulatus
urate + NADH + H+
-
-
-
?
1.17.1.4
xanthine + NAD+ + H2O
-
744255
Rhodobacter capsulatus CGMCC 1.3366
urate + NADH + H+
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.17.1.4
heterohexamer
an (alphabetagamma)2 heterohexameric enzyme
Rhodobacter capsulatus
1.17.1.4
More
enzyme structure comparisons, overview
Rhodobacter capsulatus
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.17.1.4
40
-
assay at
Rhodobacter capsulatus
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.17.1.4
51.6
-
t1/2 for the wild-type enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
63
-
t1/2 for the mutant Split166 enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
63.2
-
t1/2 for the mutant Split178 enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
65
-
all the XDHs exhibit a bell-shaped temperature-activity relationship with maximum activity at 40°C, the recombinant split variants are more thermostable than the wild-type. Both Split166 and Split178 maintain about 60% of maximal activity at 65°C, while the wild-type shows below 20% remaining activity after 30 min
Rhodobacter capsulatus
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.17.1.4
19
-
NAD+
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
22
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
93
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
111
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
191
-
NAD+
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
200
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.17.1.4
8.5
-
-
Rhodobacter capsulatus
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
1.17.1.4
4
11.5
activity range, bell-shaped pH-activity relationships for both the recombinant split variants
Rhodobacter capsulatus
pH Stability
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
1.17.1.4
4
11.5
both the recombinant split variants display similar bell-shaped pH-activity relationships with the optimum activity at pH 8.5 and remain stable in buffers ranging from pH 4.0-11.5 after incubation at 25°C for 24 h
Rhodobacter capsulatus
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.17.1.4
FAD
-
Rhodobacter capsulatus
1.17.1.4
molybdenum cofactor
-
Rhodobacter capsulatus
1.17.1.4
additional information
the purified wild-type XDH contains 2.80 iron, 0.94 FAD, and 0.72 Moco per (alphabeta)2 tetrameric subunit, Split178 has 2.73 iron, 0.95 FAD, and 0.70 Moco per (alphabetagamma)2 hexameric subunit, while Split166 incorporates 3.51 iron, 0.95 FAD, and 0.95 Moco per (alphabetagamma)2 hexamer
Rhodobacter capsulatus
1.17.1.4
NAD+
-
Rhodobacter capsulatus
1.17.1.4
[2Fe-2S]-center
-
Rhodobacter capsulatus
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.17.1.4
genes xdhA-C, development of a method that translates active Rhodobacter capsulatus (alphabetagamma)2 XDH by directly expressing the iron-sulfur domain, the flavin adenine dinucleotide domain and the sulfurated molybdenum domain as three separate proteins in Escherichia coli, recombinant expression of wild-type enzyme and mutant enzymes, i.e. an (alphabetagamma)2 heterohexameric enzyme and two (alphabetagamma)2 XDH variants, in Escherichia coli
Rhodobacter capsulatus
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.17.1.4
FAD
-
Rhodobacter capsulatus
1.17.1.4
molybdenum cofactor
-
Rhodobacter capsulatus
1.17.1.4
additional information
the purified wild-type XDH contains 2.80 iron, 0.94 FAD, and 0.72 Moco per (alphabeta)2 tetrameric subunit, Split178 has 2.73 iron, 0.95 FAD, and 0.70 Moco per (alphabetagamma)2 hexameric subunit, while Split166 incorporates 3.51 iron, 0.95 FAD, and 0.95 Moco per (alphabetagamma)2 hexamer
Rhodobacter capsulatus
1.17.1.4
NAD+
-
Rhodobacter capsulatus
1.17.1.4
[2Fe-2S]-center
-
Rhodobacter capsulatus
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.17.1.4
additional information
two (alphabetagamma)2 XDH variants, Split166 and Split178, are designed and constructed by splitting the small subunit (alphabeta)2 XDH at the N- and C-terminal ends of the L167-A178 peptide linking the iron-sulfur clusters and flavin adenine dinucleotide domains, respectively. Subunit composition of recombinant wild-type and split XDHsAs, overview. As for the co-substrate NAD+, mutant Split178 has a 1.07fold increased catalytic efficiency, while Split166 has a 3.8fold decreased catalytic efficiency compared to the wild-type XDH, for the substrate xanthine, the Split178 variant shows 1.21fold increased turnover number and 1.66fold increased catalytic efficiency, while the mutant Split166 shows a 4.31fold decrease in comparison to the wild-type enzyme
Rhodobacter capsulatus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.17.1.4
additional information
-
additional information
Michaelis-Menten steady-state kinetics, overview
Rhodobacter capsulatus
1.17.1.4
0.036
-
NAD+
pH 8.5, 40°C, Split166 mutant; pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
0.044
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
0.055
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
0.068
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
0.096
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.17.1.4
Fe2+
in the [2Fe-2S] center
Rhodobacter capsulatus
1.17.1.4
Molybdenum
in the molybdenum cofactor
Rhodobacter capsulatus
1.17.1.4
additional information
the purified wild-type XDH contains 2.80 iron, 0.94 FAD, and 0.72 Moco per (alphabeta)2 tetrameric subunit, Split178 has 2.73 iron, 0.95 FAD, and 0.70 Moco per (alphabetagamma)2 hexameric subunit, while Split166 incorporates 3.51 iron, 0.95 FAD, and 0.95 Moco per (alphabetagamma)2 hexamer
Rhodobacter capsulatus
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.17.1.4
xanthine + NAD+ + H2O
Rhodobacter capsulatus
-
urate + NADH + H+
-
-
?
1.17.1.4
xanthine + NAD+ + H2O
Rhodobacter capsulatus CGMCC 1.3366
-
urate + NADH + H+
-
-
?
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.17.1.4
xanthine + NAD+ + H2O
-
744255
Rhodobacter capsulatus
urate + NADH + H+
-
-
-
?
1.17.1.4
xanthine + NAD+ + H2O
-
744255
Rhodobacter capsulatus CGMCC 1.3366
urate + NADH + H+
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.17.1.4
heterohexamer
an (alphabetagamma)2 heterohexameric enzyme
Rhodobacter capsulatus
1.17.1.4
More
enzyme structure comparisons, overview
Rhodobacter capsulatus
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.17.1.4
40
-
assay at
Rhodobacter capsulatus
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.17.1.4
51.6
-
t1/2 for the wild-type enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
63
-
t1/2 for the mutant Split166 enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
63.2
-
t1/2 for the mutant Split178 enzyme, pH 8.5, 30 min
Rhodobacter capsulatus
1.17.1.4
65
-
all the XDHs exhibit a bell-shaped temperature-activity relationship with maximum activity at 40°C, the recombinant split variants are more thermostable than the wild-type. Both Split166 and Split178 maintain about 60% of maximal activity at 65°C, while the wild-type shows below 20% remaining activity after 30 min
Rhodobacter capsulatus
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.17.1.4
19
-
NAD+
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
22
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
93
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
111
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
191
-
NAD+
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
200
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.17.1.4
8.5
-
-
Rhodobacter capsulatus
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
1.17.1.4
4
11.5
activity range, bell-shaped pH-activity relationships for both the recombinant split variants
Rhodobacter capsulatus
pH Stability (protein specific)
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
1.17.1.4
4
11.5
both the recombinant split variants display similar bell-shaped pH-activity relationships with the optimum activity at pH 8.5 and remain stable in buffers ranging from pH 4.0-11.5 after incubation at 25°C for 24 h
Rhodobacter capsulatus
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.17.1.4
230
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
530
-
NAD+
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
1367
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
2520
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
3640
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
5310
-
NAD+
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.17.1.4
230
-
xanthine
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
530
-
NAD+
pH 8.5, 40°C, Split166 mutant
Rhodobacter capsulatus
1.17.1.4
1367
-
xanthine
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
2520
-
NAD+
pH 8.5, 40°C, wild-type enzyme
Rhodobacter capsulatus
1.17.1.4
3640
-
xanthine
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus
1.17.1.4
5310
-
NAD+
pH 8.5, 40°C, Split178 mutant
Rhodobacter capsulatus