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Literature summary for 1.17.1.4 extracted from

  • Wang, C.; Li, G.; Zhang, C.; Xing, X.
    Enhanced catalytic properties of novel (alphabetagamma)2 heterohexameric Rhodobacter capsulatus xanthine dehydrogenase by separate expression of the redox domains in Escherichia coli (2017), Biochem. Eng. J., 119, 1-8 .
No PubMed abstract available

Cloned(Commentary)

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

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 [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

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/ Products (Substrates)

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

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 and Products (Substrate)

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

Subunits Comment Organism
heterohexamer an (alphabetagamma)2 heterohexameric enzyme Rhodobacter capsulatus
More enzyme structure comparisons, overview Rhodobacter capsulatus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
assay at Rhodobacter capsulatus

Temperature Stability [°C]

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 [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
-
Rhodobacter capsulatus

pH Range

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 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

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 [mM/s]

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