1.17.1.4: xanthine dehydrogenase
This is an abbreviated version!
For detailed information about xanthine dehydrogenase, go to the full flat file.
Word Map on EC 1.17.1.4
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1.17.1.4
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uric
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1.2.1.37
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1.1.1.204
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allopurinol
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environmental protection
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ureide
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1.1.3.22
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medicine
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1.2.3.1
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xanthinuria
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oxypurines
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butyrophilins
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synthesis
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hypouricemic
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agriculture
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biotechnology
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analysis
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nutrition
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molecular biology
- 1.17.1.4
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uric
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1.2.1.37
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1.1.1.204
- allopurinol
- environmental protection
-
ureide
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1.1.3.22
- medicine
-
1.2.3.1
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xanthinuria
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oxypurines
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butyrophilins
- synthesis
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hypouricemic
- agriculture
- biotechnology
- analysis
- nutrition
- molecular biology
Reaction
Synonyms
AtXDH1, EC 1.1.1.204, EC 1.2.1.37, IAO1, More, NAD-xanthine dehydrogenase, PaoABC, Retinol dehydrogenase, Rosy locus protein, VvXDH, xanthine dehydrogenase, xanthine dehydrogenase-1, xanthine dehydrogenase-2, xanthine dehydrogenase/oxidase, xanthine oxidoreductase, xanthine-NAD oxidoreductase, xanthine/NAD+ oxidoreductase, xanthine:NAD+ oxidoreductase, XDH, XDH/XO, XDH1, XDH2, XdhC, XOR, YagR, YagS, YagT
ECTree
1.17.1.4 xanthine dehydrogenaseAdvanced search results
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results (Engineering
Engineering on EC 1.17.1.4 - xanthine dehydrogenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
G48D
naturally occuring mutation, drf mutant, missense mutation G143A, i.e. drf1-1 or xdh1-3
G48D/R941Q/T1061I
naturally occuring mutation, identification of 15 potential drf mutants, drf1 mutants contain missense mutations in XDH1, the mutant phenotypes cosegregate with a single missense mutation G143A. Targeted sequencing of XDH1 revealed missense mutations G2822A (resulting in R941Q) and C3182T (resulting in T1061I) in the remaining two mutants, respectively. Identification of a knockout mutant GK-049D04, i.e. xdh1-2, and of knockdown allele in SALK_148364 where a T-DNA is inserted in the 11th intron of XDH1, i.e. xdh1-1. Defense phenotypes of drf mutants, general phenotypes, overview. The loss-of-function single and double mutant lines for atrobhD and atrbohF and the eds1-2 null allele in the Col-0 background are crossed with xdh1-2 to make xdh1 rbohD and xdh1 rbohF, xdh1 eds1 double, and xdh1 rbohD rbohF triple mutant lines
R941Q
naturally occuring mutation, drf mutant, missense mutation G2822A, i.e. drf1-2 or xdh1-4
T1061I
naturally occuring mutation, drf mutant, missense mutation C3182T, i.e. drf1-3 or xdh1-5
G48D
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naturally occuring mutation, drf mutant, missense mutation G143A, i.e. drf1-1 or xdh1-3
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G48D/R941Q/T1061I
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naturally occuring mutation, identification of 15 potential drf mutants, drf1 mutants contain missense mutations in XDH1, the mutant phenotypes cosegregate with a single missense mutation G143A. Targeted sequencing of XDH1 revealed missense mutations G2822A (resulting in R941Q) and C3182T (resulting in T1061I) in the remaining two mutants, respectively. Identification of a knockout mutant GK-049D04, i.e. xdh1-2, and of knockdown allele in SALK_148364 where a T-DNA is inserted in the 11th intron of XDH1, i.e. xdh1-1. Defense phenotypes of drf mutants, general phenotypes, overview. The loss-of-function single and double mutant lines for atrobhD and atrbohF and the eds1-2 null allele in the Col-0 background are crossed with xdh1-2 to make xdh1 rbohD and xdh1 rbohF, xdh1 eds1 double, and xdh1 rbohD rbohF triple mutant lines
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R941Q
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naturally occuring mutation, drf mutant, missense mutation G2822A, i.e. drf1-2 or xdh1-4
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T1061I
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naturally occuring mutation, drf mutant, missense mutation C3182T, i.e. drf1-3 or xdh1-5
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E89K
G1011E
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within the molybdenum domain, no activity without oxidative activation
R40K
mutation in subunit PaoC, strong decrease in activity
R440H
mutation in subunit PaoC, strong decrease in activity, crystallization data
E803V
R881M
C535A
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resistant to conversion from dehydrogenase to oxidase by incubation with 4,4-dithiodipyridine
C535A/C992R
C535A/C992R/C1316S
C535A/C992R/C1324S
C992R
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resistant to conversion from dehydrogenase to oxidase by incubation with 4,4-dithiodipyridine
W335A/F336L
mutant oxidoreductase displaying xanthine oxidase activity
C44A/C47A
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site-directed mutagenesis, an instable subunit A mutant that cannot be purified
E220R/D517R
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site-directed mutagenesis, a subunit B mutant that is mainly dimeris incontrast to the tetrameric wild-type enzyme, inactive mutant
E232A
E232Q
site-directed mutagenesis, kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, the mutant exhibits a 12fold decrease in kred, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme
E730A
EB232Q
catalytically inactive active site mutant, inactive desulfo enzyme form
Q102A
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site-directed mutagenesis, a subunit A mutant that shows altered metal content and reduced KM and Kcat with xanthine compared to the wild-type enzyme
Q102G
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site-directed mutagenesis, a subunit A mutant that shows altered metal content and reduced KM and Kcat with xanthine compared to the wild-type enzyme
Q179A
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crystal structure determination and analysis, comparison with wild-type enzyme structure, a similar acidic pK for the wild-type and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. Oxidized wild-type and mutant Q179A reveal a similar Mo(VI) ion with each one molybdopterin, Mo=O, Mo-O-, and Mo=S ligand, and a weak Mo-O(E730) bond at alkaline pH
R135C
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mutation corresponding to human protein variant of a patient suffering from xanthinuria I. Mutation results in an active (alphabeta)2 heterotetrameric form besides an inactive alphabeta heterodimeric form missing the FeSI center
R310K
R310M
R330M
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the activity with substrate 2-hydroxy-6-methylpurine is only slightly affected
D430H
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) slightly increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Km (xanthine) slightly decreased compared to wild-type, Vmax (xanthine) slightly decreased to wild-type
D431A
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) slightly increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) increased compared to wild-type, Km (xanthine) slightly increased compared to wild-type, Vmax (xanthine) increased to wild-type
G47A
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Km and Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) increased compared to wild-type, Km and Vmax (xanthine) slightly increased compared to wild-type
K1230A
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) 2.5fold increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Km (xanthine) 2fold increased compared to wild-type, Vmax (xanthine) 2fold decreased to wild-type
N352A
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) slightly increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) slightly decreased compared to wild-type, Km (xanthine) slightly increased compared to wild-type, Vmax (xanthine) slightly decreased compared to wild-type
R427E
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Km (xanthine) slightly increased compared to wild-type, Vmax (xanthine) comparable to wild-type
S1227A
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) 2.5fold increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Km (xanthine) 2fold increased compared to wild-type, Vmax (xanthine) 2fold decreased to wild-type
S360P
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Km (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) increased compared to wild-type, Vmax (cyanoacetylhydrazone 2-formylquinoxaline-1,4-dioxide) decreased compared to wild-type, Km (xanthine) 3fold increased compared to wild-type, Vmax (xanthine) 3fold decreased compared to wild-type
additional information
E89K
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natural mutant strain, lacking iron-sulfur centers, activity to xanthine/NAD+ or xanthine/pterin not affected, but xanthine/phenazine methosulfate activity abolished
E803V
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almost complete loss of activity with hypoxanthine, weak activity with xanthine, significant aldehyde oxidase activity
E803V
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very low steady-state activity towards xanthine or hypoxanthine, loss of hydrogen bonding with one of these residues greatly influences the electron transfer process to the molybdenum center, changing the rate-limiting step in the reductive half-reaction
R881M
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almost complete loss of activity with xanthine, weak activity with hypoxanthine, significant aldehyde oxidase activity
R881M
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very low steady-state activity towards xanthine or hypoxanthine, loss of hydrogen bonding with one of these residues greatly influences the electron transfer process to the molybdenum center, changing the rate-limiting step in the reductive half-reaction
C535A/C992R
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slow conversion from dehydrogenase to oxidase by incubation with 4,4-dithiodipyridine, conversion is blocked by NADH
C535A/C992R
site-directed mutagenesis, the mutant activity in the presence of sulfhydryl residue modifiers is very low
C535A/C992R/C1316S
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completely resistant to conversion from dehydrogenase to oxidase by incubation with 4,4-dithiodipyridine
C535A/C992R/C1316S
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mutation in residues involved in conversion of xanthin dehydrogenase to xanthine oxidase by formation of disulfide bonds. Using guanidine-HCl, the mutant can be converted into the oxidase form
C535A/C992R/C1316S
site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all
C535A/C992R/C1324S
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completely resistant to conversion from dehydrogenase to oxidase by incubation with 4,4-dithiodipyridine
C535A/C992R/C1324S
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an XDH-locked enzyme mutant that cannot be induced by sulfhydryl reagents to adopt the XO form
C535A/C992R/C1324S
site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all
E232A
site-directed mutagenesis, the mutant exhibits a 12fold decrease in kred compared to wild-type
E730A
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crystal structure determination and analysis, comparison with wild-type enzyme structure, the sulfido is replaced with an oxo ligand in the inactive E730A mutant, further showing another oxo and one Mo-OH ligand at Mo, which are independent of pH
R310K
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absorption spectra similar to wild-type. 20fold decrease of kred-value
R310K
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kred, the limiting rate of enzyme reduction by substrate at high substrate concentration is 20-fold decreased
R310M
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absorption spectra similar to wild-type. 20000fold decrease of kred-value
R310M
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kred, the limiting rate of enzyme reduction by substrate at high substrate concentration is 20000-fold decreased
additional information
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T-DNA insertion mutant, loss of superoxide producing activity
additional information
generation of XDH-knockdown mutants, analysis of compromised drought-stress responses of proline biosynthesis in Arabidopsis thaliana XDH-knockdown mutants, phenotype, overview
additional information
the powdery mildew fungus Golovinomyces cichoracearum triggers defense responses in Arabidopsis mediated by the R gene RPW8.2. In a screen for mutants defective in RPW8.2-related resistance to powdery mildew, three plants with point mutations in xanthine dehydrogenase 1 (XDH1), including two that alter residues strictly conserved among xanthine dehydrogenases. The mutants show impaired resistance to powdery mildew and accumulate less H2O2 in the haustorial complex in epidermal cells invaded by the fungus. These point mutations decrease the activity of recombinant XDH1 proteins, in terms of both dehydrogenase activity and ROS production
additional information
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generation of XDH-knockdown mutants, analysis of compromised drought-stress responses of proline biosynthesis in Arabidopsis thaliana XDH-knockdown mutants, phenotype, overview
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additional information
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the powdery mildew fungus Golovinomyces cichoracearum triggers defense responses in Arabidopsis mediated by the R gene RPW8.2. In a screen for mutants defective in RPW8.2-related resistance to powdery mildew, three plants with point mutations in xanthine dehydrogenase 1 (XDH1), including two that alter residues strictly conserved among xanthine dehydrogenases. The mutants show impaired resistance to powdery mildew and accumulate less H2O2 in the haustorial complex in epidermal cells invaded by the fungus. These point mutations decrease the activity of recombinant XDH1 proteins, in terms of both dehydrogenase activity and ROS production
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additional information
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isolation of selD and xdh in-frame deletion mutants with null phenotype for biofilm formation. The wild-type strain produces significant levels of superoxide, whereas the selD and xdh mutants do not exhibit superoxide production, overview
additional information
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enzyme null mutant mice demonstrate 50% reduction in adipose mass compared to control, while obese mice exhibit increased concentrations of xanthine oxidoreductase mRNA and urate in adipose tissues. In vitro, knockdown of xanthine oxidoreductase inhibits adipogenesis and nuclear receptor PPARgamma activity
additional information
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xanthine oxidoreductase expression is elevated 2-fold in white adipose tissue of obese ob/ob mice relative to wild-type, and treatment of ob/ob mice with leptin reduces xanthine oxidoreductase mRNA to wild-type levels. Similarly, serum uric-acid levels are elevated in ob/ob mice relative to wild-type and normalized by leptin treatment. Adipose stores from 2-week-old mice lacking xanthine oxidoreductase activityshow a 12% reduction in body weight compared with wild-type, due to a 50% reduction in adipose content. Serum analysis in xanthine oxidoreductase -/- mice shows significantly decreased free fatty-acid concentrations, while no significant differences are evident for glucose and serum triglycerides
additional information
construction of a variant of the rat liver enzyme that lacks the C-terminal amino acids 1316-1331. The mutant enzymes appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified mutant protein retains about 50-70% of oxidase activity even after prolonged dithiothreitol treatment. The C-terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. But after 15 min of incubation with a high concentration of NADH, the corresponding X-ray structures show a dehydrogenase-type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the mutant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide
additional information
pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline), electron transfer from the hypoxanthine (HX)-reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on indium tin oxide (ITO) electrodes, when the polymer is present. Depending on solution pH, different processes can be identified. Not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Substrate-dependent catalytic currents can be obtained in acidic and neutral solutions, although the highest activity of XDH with natural reaction partners is in the alkaline region. Operation of the enzyme electrode without addition of the natural cofactor of XDH is feasible. Macroporous ITO electrodes are used as an immobilization platform for the fabrication of HX-sensitive electrodes. The efficient polymer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in good processability, stability, and defined signal transfer in the presence of a substrate. Method development and evaluation, overview
additional information
succesfull mechanism-based metabolic engineering of Escherichia coli strain BL21(DE3) cell factory for production of functionally active, highly-producing xanthine dehydrogenase by co-overexpression of enzyme XDH from Rhodobacter capsulatus with three global regulators (IscS, TusA and NarJ) and four chaperone proteins (DsbA, DsbB, NifS and XdhC) to aid the formation and ordered assembly of three redox center cofactors of Rhodobacter capsulatus XDH in Escherichia coli. NifS is a cysteine desulfurase, which catalyzes the sulfur transfer from L-cysteine to Moco to form Mo-S bond. Chaperone XDHC binds stoichiometric amount of Moco as a scaffold protein, interacts with NifS for the sulfuration of Moco, protects sulfurated Moco from oxidation, and further transfers to XDH, method devlopment, overview. Three helper proteins, NifS, IscS and DsbB improve the specific activity of RcXDH significantly by 30%, 94% and 49%, respectively. The combination of NifS and IscS synergistically increases the specific activity by 1.29fold, and enhances the total enzyme activity by an impressive 3.9fold
additional information
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the enzyme mutants show alterations in the Mo site structure, which changes in a pH range of 5-10, and in the influence of amino acids (Glu730 and Gln179) close to molybdenum cofactor in wild-type, and Q179A and E730A mutants, enzyme kinetics and quantum chemical studies, overview
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
additional information
Rhodobacter capsulatus CGMCC 1.3366
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succesfull mechanism-based metabolic engineering of Escherichia coli strain BL21(DE3) cell factory for production of functionally active, highly-producing xanthine dehydrogenase by co-overexpression of enzyme XDH from Rhodobacter capsulatus with three global regulators (IscS, TusA and NarJ) and four chaperone proteins (DsbA, DsbB, NifS and XdhC) to aid the formation and ordered assembly of three redox center cofactors of Rhodobacter capsulatus XDH in Escherichia coli. NifS is a cysteine desulfurase, which catalyzes the sulfur transfer from L-cysteine to Moco to form Mo-S bond. Chaperone XDHC binds stoichiometric amount of Moco as a scaffold protein, interacts with NifS for the sulfuration of Moco, protects sulfurated Moco from oxidation, and further transfers to XDH, method devlopment, overview. Three helper proteins, NifS, IscS and DsbB improve the specific activity of RcXDH significantly by 30%, 94% and 49%, respectively. The combination of NifS and IscS synergistically increases the specific activity by 1.29fold, and enhances the total enzyme activity by an impressive 3.9fold
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additional information
Rhodobacter capsulatus CGMCC 1.3366
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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
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