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4.1.99.3: deoxyribodipyrimidine photo-lyase

This is an abbreviated version!
For detailed information about deoxyribodipyrimidine photo-lyase, go to the full flat file.

Word Map on EC 4.1.99.3

Reaction

cyclobutadipyrimidine (in DNA)
= 2 pyrimidine residues (in DNA)

Synonyms

AMV025, AnPL, AtCry1, AtCry3, AtPL, BcCRY1, BcCRY2, BCIN_05g08060, BCIN_09g01620, BsUvrC, Cc-phr2, CcPL, CDP photolyase, class I CPD photolyase, class I cyclobutane pyrimidine dimer photolyase, class I photolyase, class I PL, class II AtPL, class II CPD photolyase, class II CPD-DNA photolyase, class II CPD-photolyase, class II cyclobutane pyrimidine dimer photolyase, class II DmPL, class II DNA photolyase, class II photolyase, class II PL, class III PL, cold-adapted DNA photolyase, CPD class I photolyase, CPD photolyase, CPD photolyase photolyase, CPD specific photolyase, CPD-class I photolyase, CPD-photolyase, CPD-Phr, CPD-specific DNA photolyase, CPD1, CPD2PHR, CPDI, CPDII, CPDphr, CPH1, CPH1-PHR, CpPL, Cry-DASH, Cry1, Cry2, cry3, CryA, CRYD, cryptochrome 1, cryptochrome 3, CRYPTOCHROME DASH, cryptochrome-3, cryptochrome-DASH, CsPHR, cyclobutane pyrimidine dimer photolyase, cyclobutane pyrimidine dimer PHR, cyclobutane pyrimidine dimer specific photolyase, cyclobutane pyrimidine dimer-class I photolyase, cyclobutane pyrimidine dimer-specific DNA photolyase, cyclobutane pyrimidine dimers photolyase, CYME_CMA044C, cytochrome DASH, DASH cryptochrome, deoxyribodipyrimidine photolyase, deoxyribodipyrimidine photolyase type I enzyme, deoxyribonucleate pyrimidine dimer lyase (photosensitive), deoxyribonucleic cyclobutane dipyrimidine photolyase, deoxyribonucleic photolyase, dipyrimidine photolyase (photosensitive), DmPL, DNA cyclobutane dipyrimidine photolyase, DNA photolyase, DNA photolyase type I enzyme, DNA repair protein photolyase, DNA-photoreactivating enzyme, EcPL, eukaryotic Class II CPD PHR, eukaryotic class II cyclobutane pyrimidine dimer photolyase, lyase, deoxyribonucleate pyrimidine dimer, MmCPDII, MmPL, MM_0852, photolyase, photolyase I, photolyase orthologue, photoreactivating enzyme, PHR, phr A photolyase, PHR1, PHR2, phrA, PhrB, PhrB orthologue, PhrB photolyase, PL, PRE, primase, Saci_1227, ssDNA, ssDNA AtCRY3, ssDNA photolyase, ssDNA PL, SsPL, St-photolyase, tetur12g04440, tetur12g04460, tetur35g00010, tetur35g00030, thymine dimer by photolyase, VcCry1, VcPHR, Ver3Phr, VPA1471, XlCry-DASH

ECTree

     4 Lyases
         4.1 Carbon-carbon lyases
             4.1.99 Other carbon-carbon lyases
                4.1.99.3 deoxyribodipyrimidine photo-lyase

Engineering

Engineering on EC 4.1.99.3 - deoxyribodipyrimidine photo-lyase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
W356F
-
photobleaching of 5,10-methenyltetrahydrofolate under UV-A irradiation is strongly reduced in the mutant compared with wild-type. The amount of 5,10-methenyltetrahydrofolate after UV-A irradiation is reduced by 64% for wild-type and by 20% for the mutant. Increase in the amount of oxidized FAD under UV-A irradiation due to electron donation to 5,10-methenyltetrahydrofolate by residual FADH- but a lack of photoreduction of the flavin caused by the interrupted tryptophan triad
W432F
-
photobleaching of 5,10-methenyltetrahydrofolate under UV-A irradiation is strongly reduced in the mutant compared with wild-type
R350A
-
conserved arginine forms a salt bridge with phosphate+1. Among several active-site mutants this R/A mutant is found to exhibit the greatest effect by lowering the selectivity toward cyclobutane-pyrimidine dimer-containing DNA 32-fold and the quantum yield of CPD cleavage from 98% to about 60%
W395R
deltaphrA
-
Rhodobacter sphaeroides mutant cells lacking the PhrA gene show a significant reduced survival rate after UV-illumination
Q336H
site-directed mutagenesis of the alpha13 conserved amino acid residue
E109A
E109D
-
mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
E109Q
-
mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
E274A
L375H
-
binds methenyltetrahydrofolate more weakly than wild-type enzyme
M345A
N108L
N341A
-
life-time of 2.9 ns compared to 1.3 ns for the wild-type enzyme
N378C
N378D
site-directed mutagenesis, the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD- radical (formed by electron transfer from the tryptophan chain) in plant cryptochromes (CRYs). But the mutant protein does not show this protonation. EcPL mutant protein approaches the flavin with similar kinetics to that of the aspartic acid at the corresponding position in plant CRY, but is unable to fully transfer the proton to N5 of the flavin, resulting in a FAD radical with unusual spectral properties. Possibly, the pKa values of FADH radical and/or this aspartic acid in the EcPL N378D mutant protein differ from those in native plant CRY, such that proton transfer is energetically disfavored. Absorption kinetics compared to wild-type
N378S
R226A
R342A
W277E
-
the binding affinity for CPD substrate is lower for 1000fold, although the photochemical properties and the quantum yields for catalyses (under the irradiation wavelengths at 366nm and 384 nm) of the mutant is indistinguishable from the wild-type enzyme
W277R
-
the binding affinity for CPD substrate is lower for 300fold, although the photochemical properties and the quantum yields for catalyses (under the irradiation wavelengths at 366nm and 384 nm) of the mutant is indistinguishable from the wild-type enzyme
W306F
N403A
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
N403L
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
W360F
mutation of the medial tryptophan, W360, gives a 22fold decrease of photoreduction activity relative to the wild type enzyme, no major build-up of the semiquinoid FADH radical species can be observed for W360F
W381F
the mutation causes complete loss of photoreduction activity and a loss of 70% of incorporation of the catalytic FAD compared to the wild-type enzyme
N403A
-
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
-
N403L
-
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
-
W360F
-
mutation of the medial tryptophan, W360, gives a 22fold decrease of photoreduction activity relative to the wild type enzyme, no major build-up of the semiquinoid FADH radical species can be observed for W360F
-
W381F
-
the mutation causes complete loss of photoreduction activity and a loss of 70% of incorporation of the catalytic FAD compared to the wild-type enzyme
-
Q126R
the mutation leads to a reduction of enzymatic activity
Q296H
the mutation leads to a reduction of enzymatic activity
S267A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S494A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S504A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S5A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S7A
site-directed mutagenesis, the mutant enzyme is not phosphorylated
S84A
site-directed mutagenesis, the mutant enzyme is phosphorylated
T115A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S267A
-
site-directed mutagenesis, the mutant enzyme is phosphorylated
-
S504A
-
site-directed mutagenesis, the mutant enzyme is phosphorylated
-
S5A
-
site-directed mutagenesis, the mutant enzyme is phosphorylated
-
S84A
-
site-directed mutagenesis, the mutant enzyme is phosphorylated
-
T115A
-
site-directed mutagenesis, the mutant enzyme is phosphorylated
-
A385S
-
is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Shows enhanced semiquinone stability
E283A
-
the mutation impairs enzyme activity by diminishing the quantum yield for the repair reaction by 60%
G389N
-
is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 3 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH
M353Q
-
is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Has little impact on semiquinone stability
R350A
-
the mutation demonstrates a 60% decrease in quantum yield, which indicates that Arg350 plays a key role in stabilizing the dimer
W392Y
-
is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 2 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH
R46E
R46E mutant which lacks a conserved arginine in the binding site for the antenna chromophore shows no flavin-mononucleotide and discloses an eightfold lower activity at 450 nm (blue light) wheras at 370 nm (UV-A light) its activity is only three times lower than wildtype enzyme
H354A
-
affinity to substrate is comparable to wild-type enzyme, no activity
H358A
-
affinity to substrate is comparable to wild-type enzyme, no activity
L355A
-
low affinity to the substrate
Q288A
-
affinity to substrate is comparable to wild-type enzyme
W291A
-
affinity to substrate is comparable to wild-type enzyme, mutant retains some activity
W398A
-
affinity to substrate is comparable to wild-type enzyme, mutant retains some activity
additional information