Cloned (Comment) | Organism |
---|---|
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 |
Protein Variants | Comment | Organism |
---|---|---|
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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten steady-state kinetics, overview | Rhodobacter capsulatus | |
0.036 | - |
NAD+ | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus | |
0.036 | - |
NAD+ | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus | |
0.044 | - |
NAD+ | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
0.055 | - |
xanthine | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus | |
0.068 | - |
xanthine | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
0.096 | - |
xanthine | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | in the [2Fe-2S] center | Rhodobacter capsulatus | |
Molybdenum | in the molybdenum cofactor | Rhodobacter capsulatus | |
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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
xanthine + NAD+ + H2O | Rhodobacter capsulatus | - |
urate + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | Rhodobacter capsulatus CGMCC 1.3366 | - |
urate + NADH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodobacter capsulatus | O54050 AND O54051 AND Q9X7K2 | small and large subunits encoded by genes xdhA and xdhB, and chaperone encoded by gene xdhC | - |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
xanthine + NAD+ + H2O | - |
Rhodobacter capsulatus | urate + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | - |
Rhodobacter capsulatus CGMCC 1.3366 | urate + NADH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
heterohexamer | an (alphabetagamma)2 heterohexameric enzyme | Rhodobacter capsulatus |
More | enzyme structure comparisons, overview | Rhodobacter capsulatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
assay at | Rhodobacter capsulatus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
51.6 | - |
t1/2 for the wild-type enzyme, pH 8.5, 30 min | Rhodobacter capsulatus |
63 | - |
t1/2 for the mutant Split166 enzyme, pH 8.5, 30 min | Rhodobacter capsulatus |
63.2 | - |
t1/2 for the mutant Split178 enzyme, pH 8.5, 30 min | Rhodobacter capsulatus |
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 Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
19 | - |
NAD+ | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus | |
22 | - |
xanthine | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus | |
93 | - |
xanthine | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
111 | - |
NAD+ | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
191 | - |
NAD+ | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus | |
200 | - |
xanthine | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
- |
Rhodobacter capsulatus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
4 | 11.5 | activity range, bell-shaped pH-activity relationships for both the recombinant split variants | Rhodobacter capsulatus |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Rhodobacter capsulatus | |
molybdenum cofactor | - |
Rhodobacter capsulatus | |
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 | |
NAD+ | - |
Rhodobacter capsulatus | |
[2Fe-2S]-center | - |
Rhodobacter capsulatus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
230 | - |
xanthine | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus | |
530 | - |
NAD+ | pH 8.5, 40°C, Split166 mutant | Rhodobacter capsulatus | |
1367 | - |
xanthine | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
2520 | - |
NAD+ | pH 8.5, 40°C, wild-type enzyme | Rhodobacter capsulatus | |
3640 | - |
xanthine | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus | |
5310 | - |
NAD+ | pH 8.5, 40°C, Split178 mutant | Rhodobacter capsulatus |