Information on EC 1.5.3.1 - sarcosine oxidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.5.3.1
-
RECOMMENDED NAME
GeneOntology No.
sarcosine oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
sarcosine + H2O + O2 = glycine + formaldehyde + H2O2
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
oxidative demethylation
-
-
redox reaction
-
-
-
-
reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
creatinine degradation I
-
-
creatinine degradation II
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glycine betaine degradation I
-
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creatinine degradation
-
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Glycine, serine and threonine metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
sarcosine:oxygen oxidoreductase (demethylating)
A flavoprotein (FAD). The flavin is both covalently and non-covalently bound in a molar ratio of 1:1.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-22-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
basonym Alcaligenes denitrificans
-
-
Manually annotated by BRENDA team
J5, J11
-
-
Manually annotated by BRENDA team
strain NS-129
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-
Manually annotated by BRENDA team
straight gram-negative rod
-
-
Manually annotated by BRENDA team
chinese hamster
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
KB210-8SY
-
-
Manually annotated by BRENDA team
KB210-8SY
-
-
Manually annotated by BRENDA team
genes TK0116 and TK0117
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
sarcosine oxidase catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool, yielding equimolar glycine, hydrogen peroxide, and formaldehyde or 5,10-methylenetetrahydrofolate
additional information
-
the recombinant alpha subunit forms a dimeric structure and behaves as an NADH dehydrogenase, while the beta subunit is a tetramer that has sarcosine oxidase and L-proline dehydrogenase activity.. The SOX complex assembles into the hetero-octameric (alphabeta)4 form and shows NADH dehydrogenase activity, structure of isolated subunit tetramers, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-proline + O2 + H2O
?
show the reaction diagram
-
less than 1% the rate of sarcosine
-
-
r
N-ethylglycine + O2 + H2O
acetaldehyde + glycine + H2O2
show the reaction diagram
-
-
-
-
?
N-methyl-DL-alanine + O2 + H2O
formaldehyde + DL-alanine + H2O2
show the reaction diagram
N-methyl-DL-valine + O2 + H2O
formaldehyde + DL-valine + H2O2
show the reaction diagram
N-methyl-L-leucine + O2 + H2O
formaldehyde + L-leucine + H2O2
show the reaction diagram
sarcosine + H2O + O2
?
show the reaction diagram
sarcosine + H2O + O2
glycine + formaldehyde + H2O2
show the reaction diagram
sarcosine + O2 + H2O
formaldehyde + glycine + H2O2
show the reaction diagram
sarcosine + O2 + H2O
glycine + formaldehyde + H2O2
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
sarcosine + H2O + O2
?
show the reaction diagram
sarcosine + H2O + O2
glycine + formaldehyde + H2O2
show the reaction diagram
sarcosine + O2 + H2O
formaldehyde + glycine + H2O2
show the reaction diagram
additional information
?
-
-
heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine to generate glycine, hydrogen peroxide and formaldehyde or 5,10-methylenetetrahydrofolate, depending on the availability of tetrahydrofolate. The amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme-substrate complex
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-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome
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-
-
flavin
tetrahydrofolate
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-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
slight
5-deaza-FAD
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competitive inhibition of flavinylation of apoenzyme
acetaldehyde
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acetate
Ag+
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strong inhibition
AgNO3
-
2 mM, 0% relative activity
Cu2+
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2 mM, 0% relative activity
diethyldicarbonate
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-
formaldehyde
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-
Hydroxylamine hydrochloride
-
-
iodoacetamide
iodoacetate
Methoxyacetate
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competitive
methylthioacetate
N-(cyclopropyl)glycin
-
suicide substrate
N-(cyclpopropyl)glycine
-
mechanism-based inhibitor, discussion of mechanism
N-bromosuccinimide
N-ethylmaleimide
o-phenanthroline
p-chloromercuribenzoate
phenylhydrazine
-
slight
Propionate
pyrrole-2-carboxylate
sodium lauryl sulfate
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2 mM, 0% relative activity
Tween 80
-
strong inhibition
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.31
2,6-dichlorophenolindophenol
-
-
1.6 - 16.5
N-Methyl-DL-alanine
6.7 - 173
N-Methyl-DL-valine
0.58 - 106
N-Methyl-L-leucine
31.2 - 730
oxygen
1.67
phenazine methosulfate
-
-
2.7
potassium ferricyanide
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-
0.01 - 50
sarcosine
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.2
N-ethylglycine
Corynebacterium sp.
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4.1
N-Methyl-DL-alanine
Paenarthrobacter ureafaciens
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-
3.4
N-Methyl-L-alanine
Corynebacterium sp.
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-
6.1 - 18.9
oxygen
0.0049 - 101
sarcosine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.1
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apoenzyme lacking FAD
1.81
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immobilized enzyme on alkylamine glass beads, pH 7.8, 37C
2.02
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immobilized enzyme on arylamine glass beads, pH 7.8, 37C
2.38
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soluble enzyme, pH 7.8, 37C
3
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apoenzyme reconstituted with 8-nor-8-chloro-FAD
4.1
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pH 8.0, 25C, mutant C315A, presence of FAD
23.2
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pH 8.0, 25C, mutant C315A, presence of 8-nor-8-chloro-FAD
27
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apoenzyme reconstituted with FAD
34.7
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native enzyme
44.8
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pH 8.0, 25C, native enzyme
additional information
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 10.5
-
activity range
6 - 10
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
37 - 40
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immobilized enzyme
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain B-0618)
Bacillus sp. (strain NS-129)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42000
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gel filtration, SDS-PAGE
44000
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gel filtration, SDS-PAGE
45000 - 48000
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gel filtration, SDS-PAGE
160000
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isolated recombinant beta-subunit, native PAGE
168000
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gel filtration
174000
185000
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gel filtration
190000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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or trimer, alpha,beta or alpha,beta2, 100000 (alpha), 55000 (beta), SDS-PAGE
heterooctamer
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4 * 54000, recombinant alpha-subunnit, + 4 * 43000, recombinant beta-subunit, SDS-PAGE, (alphabeta)4 , the recombinant alpha subunit forms a dimeric structure and behaves as an NADH dehydrogenase, while the beta subunit is a tetramer that has sarcosine oxidase and L-proline dehydrogenase activity.. The SOX complex assembles into the heterooctameric (alphabeta)4 form and shows NADH dehydrogenase activity, transmission electron microscopy and gel filtration
heterotetramer
monomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme inhibitor complexes, discussion of active site bindung determinants
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mutants K265M, K265Q, K265A, K265R, to 1.6-2.1 A resolution. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of K265 with Met or Arg. The side chain of K265M exhibits the same configuration in each molecule of K265M crystals and is nearly congruent with K265 in wild-type MSOX. The side chain of K265R is dramatically shifted compared with K265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of K265R is likely to contain a flipped-out R265 and exhibit negligible oxygen activation, similar to K265M. The 400fold higher oxygen reactivity observed with K265R is attributed to a minor flipped-in R265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. Structural water molecule 1 is strikingly absent in K265M and K265R
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R49K mutant after reconstitution with FAD, sitting drop vapor diffusion method, high salt or high PEG conditions
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recombinant protein
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unliganded enzyme, high flexibility of the active site loop is important for enzyme activity
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complexed with methylthioacetate, pyrrole 2-carboxylate and sulfite, and sarcosine-reduced enzyme. Sarcosine oxidase comprises alpha-, beta-, gamma- and delta-subunits, FAD and FMN cofactors, and a large internal cavity. Methylthioacetate and pyrrole 2-carboxylate are sandwiched between the re-face of the FAD isoalloxazine ring and the beta-subunit C-terminal residues. Reduction of flavin cofactors shifts the beta-subunit A1 residue towards the alpha-subunit M55, forming a surface cavity at the oxygen-channel vestibule and rendering the beta-subunit C-terminal residues mobile. Three channels connect the cavity and the enzyme surface. Two of them exist in the inter-subunit space between alpha- and beta-subunits, and the substrate sarcosine seems to enter the active site through either of these channels and reaches the reside of the FAD isoalloxazine ring by traversing the mobile beta-subunit C-terminal residues. The third channel goes through the alpha-subunit and has a folinic acid-binding site
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in complex with dimethylglycine and folinic acid. The alpha subunit consists of two domains, contains NAD+ and binds folinic acid. The beta subunit contains dimethylglycine, FAD and FMN. The gamma subunit is in contact with two domains of alpha subunit and has possibly a folate-binding structure. The delta subunit contains a single atom of and has a Cys3His zinc finger structure
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alpha subunit contains NAD+ and putative folate binding site. The FAD binding site is in the beta subunit, FMN is bound at the interface of the alpha and beta subunit. A zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta subunit
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
labile below
392378
6.5 - 11
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671547
6.5 - 9.5
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20C, stable for 20 h
392377
7 - 10
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392378
7 - 9
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392376
8 - 10
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purified enzyme, stable at
724775
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
pH 6.5-9.5, 20 h
30
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pH 7-9, 10 min
40
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pH 8.0, 24 h, 50% loss of activity
45
-
10 min, complete loss of activity
65
10 min, complete loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, dialyzed enzyme
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity matrix is 5-formyltetrahydrofolate coupled to AH-sepharose
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native enzyme by ammoniumm sulfate fractionation, hydrophobic interaction and anion exchange chromatography, and gel filtration
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recombinant His-tagged alpha and beta subunits from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity
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recombinant native and mutant enzyme
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recombinant protein
recombinant proteins
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recombinant proteins using His-tag
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straight gram-negative rod
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to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
all four subunits
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expression in Escherichia coli
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genes TK0116 and TK0117, separate cloning and expression in Eschericia coli strain BL21(DE3) of His-tagged alpha and beta subunits of SOX
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mutant proteins expressed as His-tag fusion proteins in Escherichia coli BL21(DE3)
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mutant proteins expressed in Escherichia coli BL21(DE3)
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native and mutant enzyme
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native and mutant enzyme in Escherichia coli BL21(DE3), R49 mutants contain His-tag
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C265A
-
almost wild type activity but much more stable against thiol modifying inhibitors
C265D
-
almost wild type activity but much more stable against thiol modifying inhibitors
C265R
-
almost wild type activity but much more stable against thiol modifying inhibitors
C265S
-
almost wild type activity but much more stable against thiol modifying inhibitors
D35A
-
D35 important for interaction with FAD
D35E
-
D35 important for interaction with FAD
D35N
-
D35 important for interaction with FAD
C314A
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mutation of the covalent attachment site of FAD, mutant forms unstable complexes with FAD. In situ reconstitution of activity by assaying mutant in presence of FAD or 8-nor-8-chloro-FAD, giving a specific activity of 14% or 80% of wild-type, resp. Mutant exhibits high affinity for reduced flavin
C315A
-
inactive, no bound FAD, forms stable non-covalent complex with 5-deazaFAD
H269N
-
His269 is probably not the active-site base but involved in interaction with substrate
H45A
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contains covalently bound FAD, 4fold less FAD than in wild type enzyme, catalytic properties similar than wild type enzyme, 50% of wild type activity
H45N
-
contains covalently bound FAD, 4fold less FAD than in wild type enzyme, catalytic properties similar than wild type enzyme, 50% of wild type activity
R49A
-
inactive, no bound FAD, forms stable non-covalent complex with 5-deazaFAD
R49Q
-
inactive
Y317F
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20fold decrease in the maxumim rate of the reductive half-reaction. Unlike wild-type, Kd-values of mutant are pH-dependent
K265A
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at least 250fold decrease in reaction rate. Crystallization analysis
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K265M
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at least 250fold decrease in reaction rate. Crystallization analysis
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K265Q
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at least 250fold decrease in reaction rate. Crystallization analysis
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K265R
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at least 250fold decrease in reaction rate. Crystallization analysis
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H173N
-
leads to catalytically inactive beta subunit, study on organization of subunits and coenzymes
K171A
-
39% activity of the wild-type enzyme, strongly increased Km for oxygen
K171D
-
32% activity of the wild-type enzyme, strongly increased Km for oxygen
K171R
-
58% activity of the wild-type enzyme
K358A
-
inactive
K358D
-
inactive
K358R
-
0.07% of activity, increased Km
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
production of soluble apoenzyme lacking FAD by controlled expression in Escherichia coli. Reconstitution of enzyme by incubation with FAD. Autoflavinylation occurs in a reaction that proceeds via reduced flavin intermediate and requires only apoenzyme and FAD
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
Show AA Sequence (2157 entries)
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