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K159M
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site-directed mutagenesis, the mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview
S114A
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site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme
S114A/Y155F
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site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme
Y155F
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site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme
Y155F/K159A
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site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme
H5Q
a naturally occuring non-synonymous single nucleotide polymorphism (SNP), rs12529 C>G or AKR1C3-2 in exon 1 of AKR1C3
R301L
site-directed mutagenesis, the mutation greatly affects the 3alpha-hydroxysteroid dehydrogenase activity towards 5alpha-dihydrotestosterone and almost completely abolishes the 17beta-hydroxysteroid dehydrogenase activity of the enzyme
R304L
site-directed mutagenesis, the mutation greatly affects the 3alpha-hydroxysteroid dehydrogenase activity towards 5alpha-dihydrotestosterone and abolishes the 17beta-hydroxysteroid dehydrogenase activity of the enzyme
C217A
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resistant to inactivation by secosteroids, therefore Cys217 is the point of covalent attachment of acetylenic ketones
D50E
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1/30th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis
D50N
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1/30th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis
E276R
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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview
H117A
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1/500th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis
K84M
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inactive, unable to bind steroids
K84R
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inactive, unable to bind steroids
N167A
site-directed mutagenesis, most impaired enzyme
Q190A
site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic cente
Q270K
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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview
Q270K/E276R
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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview
R276E
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site-directed mutagenesis, the mutant shows increased preference for the oxidation reaction compared to the wild-type enzyme
R276G
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site-directed mutagenesis, the mutant shows slightly increased preference for the reduction reaction compared to the wild-type enzyme
S166A
site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic center
Y205F
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kinetically indistinguishable from the wild type, no general amino acid for catalysis in 3alpha-hydroxysteroid dehydrogenase
Y216S
site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic cente
Y55F
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inactive, unable to perform steroid oxidoreduction, strongest candidate for the general amino acid
Y55S
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inactive, strongest candidate for the general amino acid
K159A
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site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme
K159A
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site-directed mutagenesis, the mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview
F118A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
F118A
largest changes in kcat/Km
F129A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
F129A
largest changes in kcat/Km, F129A alters the stereochemical preference from oxidizing predominantly the S,S-stereoisomer to oxidizing predominantly the R,R-stereoisomer of benzo[g]chrysene-11,12-dihydrodiol
L54A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
L54A
mutant shows intermediate changes in kcat/Km versus wild type
N306A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
N306A
mutant shows intermediate changes in kcat/Km versus wild type
R276M
elimination of salt bridge between Arg276 and the 2'-phosphate of AMP results in 100fold decrease of the affinity to NADP(H)
R276M
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site-directed mutagenesis, mutant does no longer form a salt-linkage to the phosphate of 2'-AMP, and does no longer bind tightly to NAD(P)H, the burst phase kinetics for the NADP+-dependent oxidation of 3alpha-diol is eliminated
R276M
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site-directed mutagenesis, the mutant shows slightly increased preference for the reduction reaction compared to the wild-type enzyme
T226A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
T226A
smallest change in kcat/Km is observed when alanine is used to substitute hydrophilic residues
T24A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
T24A
smallest change in kcat/Km is observed when alanine is used to substitute hydrophilic residues
W148Y
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site-directed mutagenesis, kinetically similar to wild type enzyme, no function in ligand binding
W148Y
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can catalyse steroid oxidoreduction similar to wild type, plays no role in steroid binding or catalysis
W227A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, 23fold decreased affinity for progesterone, effects of mutation on binding constants and kinetics, overview
W227A
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site-directed mutagenesis, highly reduced activity compared to the wild-type, very slow chemical transformation
W227A
largest changes in kcat/Km
W227Y
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site-directed mutagenesis, important role in binding steroid hormones, but not small substrates or inhibitors, interacts with the C and/or D-rings of steroid ligand
W227Y
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mainly influenced in steroid binding
W86Y
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site-directed mutagenesis, important in binding steroids and non-steroidal anti-inflamatory drugs, region in which it resides is part of the substrate/inhibitor binding pocket, near the A-, and B-rings of bound steroid
W86Y
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plays role in cofactor and steroid binding
Y310A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview
Y310A
mutant shows intermediate changes in kcat/Km versus wild type
additional information
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co-immobilization of the enzyme with diaphorase of Clostridum sp. onto alkylamine glass beads through glutaraldehyde coupling for determination of bile acids, method opimization and evaluation, loss of 30% of activity after 4 months of regular use, overview
additional information
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construction of insertion mutants, overview
additional information
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potential CoA binding sites spanning residues 182 to 193. N-terminal region is thought to contain the NAD(P)+ binding site, the middle and C-terminal regions the active site of the enzyme
additional information
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potential CoA binding sites spanning residues 182 to 193. N-terminal region is thought to contain the NAD(P)+ binding site, the middle and C-terminal regions the active site of the enzyme
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additional information
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positions of Tyr/Lys pair are conserved across the aldo-keto reductase and short-chain dehydrogenase/reductase family. Tyr retains ability to form ternary complex and acts as general acid