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G135A
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the mutant shows decreased activity with 3-bromoacetophenone as compared to the wild type enzyme
G180A
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the mutant shows slightly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
I190A
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the mutant shows decreased activity with 3-bromoacetophenone as compared to the wild type enzyme
M186A
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the mutant shows increased activity with 3-bromoacetophenone as compared to the wild type enzyme
Q187A
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the mutant shows increased activity with 3-bromoacetophenone as compared to the wild type enzyme
V181A
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the mutant shows strongly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
W144A
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the mutant shows slightly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
Y141A
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the mutant shows slightly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
G135A
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the mutant shows decreased activity with 3-bromoacetophenone as compared to the wild type enzyme
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G180A
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the mutant shows slightly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
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Q187A
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the mutant shows increased activity with 3-bromoacetophenone as compared to the wild type enzyme
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V181A
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the mutant shows strongly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
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W144A
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the mutant shows slightly increased activity with 3-bromoacetophenone as compared to the wild type enzyme
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DELTA31
deletion of the N-terminal 31 residues, kinetic parameters KM and kcat are essentially the same as those of the wild-type. It forms a tetramer in solution, which is similar to the wild-type but is less stable. Melting temperature is 48°C, which is lower than that of the wild-type (52°C)
H68D
produces (R)-enantiomer with low optical purity and yield
H68D/P69D
produces (R)-enantiomer with low optical purity and yield
P69D
produces (R)-enantiomer with low optical purity and yield
S67D
produces (R)-enantiomer with low optical purity and yield
S67D/P69D
produces (R)-enantiomer with low optical purity and yield
D218V
J9P7P2
single nucleotide polymorphism
W230P
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exhibits properties similar to the parent enzyme with regard to steroid specificities and kinetics toward substrates
Y172A
Km value for 9,10-phenanthrenequinone (model substrate) is 12.5 times higher than that for the wild-type enzyme
C227S
displays a similar kcat, but a 30fold higher Km value for S-nitrosoglutathione, and does not show substrate inhibition
D236A/K238P/D239K/S240A/I241T/R242K/T243S/V244P
mutations introduce activity towards S-nitrosoglutathione
P230W/D236A/K238P/D239K/S240A/I241T/R242K/T243S/V244P
mutations introduce activity towards S-nitrosoglutathione
Q142M/C143S/P230W/D236A/K238P/D239K/S240A/I241T/R242K/T243S/V244P/H270S
mutations introduce activity towards S-nitrosoglutathione
V88I
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functional genetic polymorphism. Mutation results in CBR1 isoforms with different binding affinities for cofactor NADPH and inhibitor rutin as well as different maximal velocities for reaction with daunorubicin and prostaglandin E2
V166A
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the mutant has increased activity with 2,2,2-trifluoroacetophenone compared to the wild type enzyme
W230P
exhibits properties similar to the parent enzyme with regard to steroid specificities and kinetics toward substrates
A89N/F154Y
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Vmax and kcat values of the mutant enzyme are enhanced by 2.08 and 3.86fold, respectively, while the Km value decreases by 2.36fold compared to the wild type enzyme
Q245H
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inversion of enantioselectivity from (R)- to (S)-configuration of reduced acetophenone product, with enhanced enantioselectivity
Q245L
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inversion of enantioselectivity from (R)- to (S)-configuration of reduced acetophenone product, with enhanced enantioselectivity
Q245P
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inversion of enantioselectivity from (R)- to (S)-configuration of reduced acetophenone product, with enhanced enantioselectivity
S41A/S42A/S43Q/W63I/Y64D/N65I/S66N
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site-sirected mutagenesis, mutant S1M1 shows reduced activity and inverted cofactor specificity, using exclusively NADH, compared to the wild-type enzyme
S41A/S42A/S43R/W63I/Y64D/N65I/S66N
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site-sirected mutagenesis, mutant S1M4 shows reduced activity and inverted cofactor specificity, using exclusively NADH, compared to the wild-type enzyme
S41A/S42A/S43Q/W63I/Y64D/N65I/S66N
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site-sirected mutagenesis, mutant S1M1 shows reduced activity and inverted cofactor specificity, using exclusively NADH, compared to the wild-type enzyme
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S41A/S42A/S43R/W63I/Y64D/N65I/S66N
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site-sirected mutagenesis, mutant S1M4 shows reduced activity and inverted cofactor specificity, using exclusively NADH, compared to the wild-type enzyme
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E142M
increase in reaction velocity
E142V
increase in reaction velocity
S172A
site-directed mutagenesis, decrease of melting temperature compared to wild-type. No catalytic activity
S172A
melting temperature is 46°C, which is lower than that of the wild-type (52°C). Shows no activity
S172T
site-directed mutagenesis, decrease of melting temperature compared to wild-type. Catalytic activity similar to wild-type
S172T
melting temperature is 48°C, which is lower than that of the wild-type (52°C). Activity is essentially the same as the wild-type
S67D/H68D
site-directed mutagenesis, about 10fold increase and 20fold decrease for NADH and NADPH kcat/KM-value, respectively
S67D/H68D
double-point mutation inside the coenzyme-binding pocket results in a nearly 10fold increase and a 20fold decrease in the kcat/KM value when NADH and NADPH are used as cofactors, respectively, with kcat remaining essentially the same. Shows similar thermal stability to wild-type
S67D/H68D
produces (R)-1-phenyl-1,2-ethanediol with high optical purity of 95.4% and a yield of 83.1% in the NADH-linked reaction. It results in a nearly 10fold increase and a 20fold decrease in the kcat/Km value when NADH and NADPH are used as the cofactors, respectively, but maintaining a kcat value essentially the same with respect to wild-type. The mutant has a stronger preference for NADH and weaker binding for NADPH. It exhibits a secondary structure and melting temperature similar to the wild-type form. NADH provides maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converts the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability
V270D
site-directed mutagenesis, catalytic acitivity similar to wild-type, mutant forms a dimer
V270D
renders the SCR as a homodimer, rather than a tetramer, without affecting the enzymatic activity. Melting temperature is 45°C, which is lower than that of the wild-type (52°C)
Y187A
site-directed mutagenesis, decrease of melting temperature compared to wild-type. No catalytic activity
Y187A
melting temperature is 45°C, which is lower than that of the wild-type (52°C). Shows no activity
Y187F
site-directed mutagenesis, decrease of melting temperature compared to wild-type. No catalytic activity
Y187F
melting temperature is 46°C, which is lower than that of the wild-type (52°C). Shows no activity
P230W
active towards isatin and 9,10-phenanthrenequinone as the wild-type
P230W
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exhibits properties similar to the parent enzyme with regard to steroid specificities and kinetics toward substrates
V244M
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naturally occuring polymorphism in isozyme CBR3, the isozyme CBR M244 may reflect a faster association-dissociation rate for NADPH/NADP+ per catalytic cycle, residue 244 is not involved in direct cofactor interaction, overview
V244M
inactive towards menadione, active towards isatin and 9,10-phenanthrenequinone as the wild-type
additional information
site-directed mutagenesis, deletion of the N-terminal 31 residues, catalytic parameters similar to wild-type, decrase in melting temperature
additional information
codon variants DELTA24 (truncation of disorder sequence of 4-27 bp at the 5'-terminus), aRCR (its nine out of 11 rare codons are adapted according to the codon bias in Escherichia coli), and mRCR (its disorder sequence of 4-27 bp at the 5'-terminus is truncated and nine out of 11 rare codons are adapted) are expressed at higher levels than the wild-type. Mutant mRCR presents highest enzyme activity. When compared with wild-type, mRCR shows an increase of 35.6% in the total activity of cell-free extracts
additional information
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codon variants DELTA24 (truncation of disorder sequence of 4-27 bp at the 5'-terminus), aRCR (its nine out of 11 rare codons are adapted according to the codon bias in Escherichia coli), and mRCR (its disorder sequence of 4-27 bp at the 5'-terminus is truncated and nine out of 11 rare codons are adapted) are expressed at higher levels than the wild-type. Mutant mRCR presents highest enzyme activity. When compared with wild-type, mRCR shows an increase of 35.6% in the total activity of cell-free extracts
additional information
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removal of the extra-loop domain in 3alpha/hydroxysteroid dehydrogenase/carbonyl reductase may lead to the formation of an enzymatically active homotetramer
additional information
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gene sniffer mutants are highly sensitive to hyperoxia-induced oxidative stress, mutations in the gene lead to a reduced life span, age-related neurodegeneration, and motoric dysfunctions that deteriorate with age, analysis of the central nervous system in sniffer mutants reveals signifcant cortex and neutrophil vacuolization and apoptosis in the brain, overexpression confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness, overview
additional information
residues of CBR3 exchanged to the corresponding amino acids of CBR1. Mutation of nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 to the corresponding residues of CBR1 reveals a much higher kcat/Km value (5.7 micromol/min) towards isatin
additional information
residues of CBR3 exchanged to the corresponding amino acids of CBR1. Mutation of nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 to the corresponding residues of CBR1 reveals a much higher kcat/Km value (5.7 micromol/min) towards isatin
additional information
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design and construction of mutant enzymes, e.g. S1M1 and S1M4, with inverted cofactor specificity using computer-aided methods, in silico screening, three-dimensional structure modeling, and site-sirected mutagenesis, mutant screening, overview, the mutant enzyme utilizes NADH, but not NADPH as cofactor
additional information
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design and construction of mutant enzymes, e.g. S1M1 and S1M4, with inverted cofactor specificity using computer-aided methods, in silico screening, three-dimensional structure modeling, and site-sirected mutagenesis, mutant screening, overview, the mutant enzyme utilizes NADH, but not NADPH as cofactor
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additional information
mutation of C-terminal peroxisomal targeting sequence SRL to SKL, SHL, SLL, and SL. Mutants SLL and SL are not catalytically active. Mutant SHL is gradually inactivated during incubation at 0°C. Both mutants SHL and SKL are tetramers and show kinetic parameters similar to wild-type
additional information
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mutation of C-terminal peroxisomal targeting sequence SRL to SKL, SHL, SLL, and SL. Mutants SLL and SL are not catalytically active. Mutant SHL is gradually inactivated during incubation at 0°C. Both mutants SHL and SKL are tetramers and show kinetic parameters similar to wild-type