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D132G
site-directed mutagenesis, the mutant enzyme kinetically resembles isozyme AtCM1, it retains activation by tryptophan, although to a lesser extent than observed with wild-type AtCM3
G149A
site-directed mutagenesis, the mutation eliminates the effector action of both phenylalanine and tyrosine
G149D
site-directed mutagenesis, the mutation eliminates the effector action of both phenylalanine and tyrosine
G213A
site-directed mutagenesis, mutation in effector binding site, the mutation eliminates the effect of aromatic amino acids on enzymatic activity
G213P
site-directed mutagenesis, mutation in effector binding site, the mutation eliminates the effect of aromatic amino acids on enzymatic activity
H145Q
site-directed mutagenesis, mutation in effector binding site, the mutation has varyring effects on the EC50 values for the aromatic amino acid effectors but does not change either positive or negative effects on enzymatic activity
R79k
site-directed mutagenesis, mutation in effector binding site, the mutation has varyring effects on the EC50 values for the aromatic amino acid effectors but does not change either positive or negative effects on enzymatic activity
V217T
site-directed mutagenesis, mutation in effector binding site
D233I
Q9Y7B2
little reduced regulatory range through tyrosine and tryptophan than wild-type enzyme
D233T
Q9Y7B2
strong reduced regulatory range through tyrosine and tryptophan than wild-type enzyme
C75S
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viscosity-insensitive
C88K/R90S
lower activity than wild-type enzyme
C88S/R90K
lower activity than wild-type enzyme
DELTA118-127
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large increase in KM and slower turnover relative to wild-type enzyme
DELTA118-127/K111N/A112S/V113N
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large increase in KM and slower turnover relative to wild-type enzyme
DELTA118-127/R116L/P117T
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large increase in KM and slower turnover relative to wild-type enzyme
DELTA119-127/D118N
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large increase in KM and slower turnover relative to wild-type enzyme
R90A
no activity detectable
R90K
The ES, TS, and product structures of the mutants are determined based on the wild-type structure. The hydrogen-bond lengths of the mutants differ from the wild-type. The two mutants chemical reaction progresses in a similar way. No large geometrical changes in and around the active site along the reaction path: only a small rearrangement of the hydrogen-bond sites. As for Lys90/Cit90 mutant reactions, no large conformational change is observed in the overall protein structure except for the geometries around the mutation point. Although the catalytic activity of R90K is inferior to that of the wild-type, the enzymatic mechanism of the R90K mutant is similar to the wild-type. The main anticatalytic factor of R90Cit mutant is the ES stabilization as a result of destabilizing the substrate by the surrounding electrostatic field because of the mutated enzyme. Therefore the TS stabilization mechanism of the Cit90 mutant is quite different from that of the wild-type BsCM
R90Q
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complete inactivation of enzyme
A32S
increased catalytic efficiency
DELTA102-285
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hybrid of chorismate mutase and allosteric domain from P-protein without prephrenate dehydratase
H131A
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30% activity compared to wild-type enzyme
H153N
-
lower turnover and higher KM than wild-type enzyme
H189N
-
much lower turnover than wild-type enzyme
H197N
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lower turnover and higher KM than wild-type enzyme
H239N
-
lower turnover and higher KM than wild-type enzyme
H245N
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lower turnover and higher KM than wild-type enzyme
H257A
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lower turnover and higher KM than wild-type enzyme
H265A
-
lower turnover and higher KM than wild-type enzyme
H347N
-
lower turnover than wild-type enzyme
I81L/V85I
reduced catalytic efficiency, alters packing against the hydrophobic ring face of the reacting molecule
K37A
-
more poorly expressed than wild-type, inactive and instable
K39N
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the mutant enzymes Lys39Arg, Lys39Asn, Lys39Gln, Gln88Arg, and Gln88Glu show similar structures to the wild-type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing small deviation. The turnover numbers for the mutant enzymes Lys39Arg, Lys39Asn and Lys39Gln are 335fold, 820fold and 4090fold lower than the turnover number of the wild-type enzyme, no significant differences in Km-value for chorismate between Lys39Arg, Lys39Asn, and the wild-type enzyme
K39Q
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the mutant enzymes Lys39Arg, Lys39Asn, Lys39Gln, Gln88Arg, and Gln88Glu show similar structures to the wild-type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing small deviation. The turnover numbers for the mutant enzymes Lys39Arg, Lys39Asn and Lys39Gln are 335fold, 820fold and 4090fold lower than the turnover number of the wild-type enzyme, no significant differences in Km-value for chorismate between Lys39Arg, Lys39Asn, and the wild-type enzyme
K39R
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the mutant enzymes Lys39Arg, Lys39Asn, Lys39Gln, Gln88Arg, and Gln88Glu show similar structures to the wild-type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing small deviation. The turnover numbers for the mutant enzymes Lys39Arg, Lys39Asn and Lys39Gln are 335fold, 820fold and 4090fold lower than the turnover number of the wild-type enzyme, no significant differences in Km-value for chorismate between Lys39Arg, Lys39Asn, and the wild-type enzyme
L7I
no significant effect on catalytic efficiency
Q88R
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the mutant enzymes Lys39Arg, Lys39Asn, Lys39Gln, Gln88Arg, and Gln88Glu show similar structures to the wild-type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing small deviation. The turnover numbers for the mutant enzymes Lys39Arg, Lys39Asn and Lys39Gln are 335fold, 820fold and 4090fold lower than the turnover number of the wild-type enzyme, no significant differences in Km-value for chorismate between Lys39Arg, Lys39Asn, and the wild-type enzyme
V35I
increased KM and turnover values compared to wild type
D48G
-
the mutation causes kcat/Km to decrease by 3 fold
F77W
-
mutant exhibits decreased activity compared to the wild type enzyme
Q88N
-
mutant exhibits decreased activity compared to the wild type enzyme
R51Q
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mutant exhibits decreased activity compared to the wild type enzyme
V35A
-
mutant exhibits decreased activity compared to the wild type enzyme
D69A
site directed mutagenesis constitute the catalytic site
E109A
site directed mutagenesis constitute the catalytic site
E109Q
site directed mutagenesis constitute the catalytic site
G86A
mutant shows reduced kcat and Km values compared to the wild type enzyme
K60A
site directed mutagenesis constitute the catalytic site
R134A
site directed mutagenesis constitute the catalytic site
R49A
site directed mutagenesis constitute the catalytic site
R72A
site directed mutagenesis constitute the catalytic site
Y105A
site directed mutagenesis constitute the catalytic site
E109A
-
site directed mutagenesis constitute the catalytic site
-
G86A
-
mutant shows reduced kcat and Km values compared to the wild type enzyme
-
R134A
-
site directed mutagenesis constitute the catalytic site
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R72A
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site directed mutagenesis constitute the catalytic site
-
Y105A
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site directed mutagenesis constitute the catalytic site
-
E23D
abolishes the substrate-induced homotrophic effect, but retains the effector-induced heterotrophic effecs, the coupling between helix 11 and helix 12 is weakened
I225T
-
mutant enzyme Ile225Thr is activated by Trp, but is insensitive to Tyr
N139L/R156L
increase in global flexibility
T226D
-
reduced regulatory range through tyrosine and tryptophan than wild-type enzyme
Y234F
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enzymes with mutations of Tyr234, especially Tyr234Phe are unresponsive to Tyr but are activated by Trp
DELTA117-127
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large increase in KM and slower turnover relative to wild-type enzyme
DELTA117-127
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lower turnover and lower KM than wild-type enzyme
Q88E
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the mutant enzymes Lys39Arg, Lys39Asn, Lys39Gln, Gln88Arg, and Gln88Glu show similar structures to the wild-type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing small deviation. The turnover numbers for the mutant enzymes Lys39Arg, Lys39Asn and Lys39Gln are 335fold, 820fold and 4090fold lower than the turnover number of the wild-type enzyme, no significant differences in Km-value for chorismate between Lys39Arg, Lys39Asn, and the wild-type enzyme
Q88E
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mutation of Gln88 to Glu in the monofunctional chorismate mutase results in an enzyme with a pH profile of activity significantly different from that of the wild-type protein
T226I
-
mutant Thr226Ile
T226I
-
reduced regulatory range through tyrosine and tryptophan than wild-type enzyme
additional information
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CM enzymes, which are not precisely annotated in the original genome sequence of Mycobacterium tuberculosis H37Rv and the subsequent reannotation. In the annotation, two ORFs (Rv0948c and Rv1885) with some similarity to CMs, including the well-known monofunctional periplasmic CM from Erwinia herbicola, are found, although these ORFs are described as conserved hypothetical proteins
additional information
generation of the expression construct N-terminally hexahistidine-tagged AtCM1 lacking the plastid localization peptide, AtCM1DELTA66
additional information
generation of the expression construct N-terminally hexahistidine-tagged AtCM1 lacking the plastid localization peptide, AtCM1DELTA66
additional information
generation of the expression construct N-terminally hexahistidine-tagged AtCM1 lacking the plastid localization peptide, AtCM1DELTA66
additional information
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generation of the expression construct N-terminally hexahistidine-tagged AtCM1 lacking the plastid localization peptide, AtCM1DELTA66
additional information
mutant Arg90Cit, a sluggish variant of Bacillus subtilis chorismate mutase, in which a cationic active-site arginine is replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction
additional information
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mutant Arg90Cit, a sluggish variant of Bacillus subtilis chorismate mutase, in which a cationic active-site arginine is replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction
additional information
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proteins containing residues 1-285 and residues 1-300 retain full chorismate mutase activity and prephenate dehydratase activity, but exhibit no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retain full prephenate dehydratase activity, but lack mutase activity
additional information
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genetically engineered monofunctional chorismate mutase that contains only 109 amino acids starting with the bifunctional P protein that also exhibits prephenate dehydratase activity and is composed of 386 amino acids
additional information
EcCM active-site residues (Leu7, Ala32, Val35, Asp48, Ile81, Val85) that mutated in our previous computational design experiment. Each of the 114 variants tested for complementation of the chorismate mutase deficiency of the auxotrophic Escherichia coli KA12/pKIMP-UAUC system. 34% of all single mutants are scored as biological active
additional information
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EcCM active-site residues (Leu7, Ala32, Val35, Asp48, Ile81, Val85) that mutated in our previous computational design experiment. Each of the 114 variants tested for complementation of the chorismate mutase deficiency of the auxotrophic Escherichia coli KA12/pKIMP-UAUC system. 34% of all single mutants are scored as biological active
additional information
Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing encodes a truncated protein (GR-CM-1t) lacking the chorismate mutase domain with no activity
additional information
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Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing encodes a truncated protein (GR-CM-1t) lacking the chorismate mutase domain with no activity
additional information
The results of the mutagenesis and the activity show that Arg49, Lys 60, Arg72, and Arg134 are essential for catalysis
additional information
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The results of the mutagenesis and the activity show that Arg49, Lys 60, Arg72, and Arg134 are essential for catalysis
additional information
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The results of the mutagenesis and the activity show that Arg49, Lys 60, Arg72, and Arg134 are essential for catalysis
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additional information
silencing of gene Tparo7, mutant phenotype, plant rhizosphere colonization tests on tomato plants, detailed overview
additional information
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silencing of gene Tparo7, mutant phenotype, plant rhizosphere colonization tests on tomato plants, detailed overview
additional information
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silencing of gene Tparo7, mutant phenotype, plant rhizosphere colonization tests on tomato plants, detailed overview
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