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analysis
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a L-lactate-selective microbial biosensor is developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing FCb2. The HpCYB2 gene, encoding FCb2, under the control of the strong Hansenula polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration is transformed to the recipient strain Hansenula polymorpha C-105 (gcr1 catX) impaired in glucose repression and devoid of catalase activity. The biosensor based on recombinant yeast cells exhibit a higher Km value (Km: 3.02 mM) and hence expanded linear range toward l-lactate as compared to a similar sensor based on the initial cells of Hansenula polymorpha C-105 (Km: 0.33 mM)
A198G
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turnover reduced to 50%
D282N
site-directed mutagenesis, while the wild-type mutant has residue R289 in a distal or a proximal conformation, the mutant shows R289 only in a distal conformation
E91K
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mutation has no effect on the rate of cytochrome c reduction, no significantly different behavior with regard to inhibition by ferrocytochrome c
F52A
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mutation has no effect on the rate of cytochrome c reduction
L230A/A198G
the double mutant enzyme has a 6fold greater catalytic efficiency with L-mandelate than with L-lactate
R289K
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kcat (1/sec) (substrate: L-lactate): 8.6 (in 200 mM phosphate: 9.2, in 400 mM potassium acetate: 8.8, in 400 mM KCl: 9.2, in 400 mM KBr: 7.8), Km (mM) (substrate: L-lactate): 7.0 (in 200 mM phosphate: 8.7, in 400 mM potassium acetate: 9.2, in 400 mM KCl: 6.5, in 400 mM KBr: 5.8). Mutant is not sensitive for excess lactate concentration. In contrast to the wild-type enzyme high concentrations of acetate, phosphate, chloride and bromide show no influence on the mutant enzyme
R376K
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mutant enzyme shows no activity
R38E
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activity and inhibitory profile similar to wild type
Y254del
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deletion mutant
A198G/L230A
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double mutant, turnover reduced to less than 10%
A198G/L230A
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double mutant enzyme shows significant activity towards L-mandelate
A198G/L230A
crystallization data
A67L
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reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
A67L
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
A67Q
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reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
A67Q
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
D72A
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activity and inhibitory profile similar to wild type
D72A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
E63K
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reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
E63K
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
F39A
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reaction proceeds slower than in wild type
F39A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
H373Q
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mutant
H373Q
His373 acts as an active site base during the oxidation of lactate to pyruvate. The decrease of 3500fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most affected by the mutation. Primary deuterium and solvent kinetic isotope affects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in wild-type. Structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, shows that the orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. Active site residues Arg289, Asp292, and Leu286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively
H373Q
kcat (1/sec) (substrate:lactate): 0.031 (intact protein), 0.057 (flavin domain only), Km (mM) (substrate: lactate): 0.65 (intact protein), 0.25 (flavin domain only)
H373Q
the mutation results in a 34 orders of magnitude decrease in kcat and a slight increase in L-lactate Km
K73A
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activity and inhibitory profile similar to wild type
K73A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
L230A
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mutant
L230A
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turnover reduced to less than 10%
L230A
crystallization data
L230A
the mutant flavocytochrome b2 displays increased selectivity for (S)-2-hydroxyoctanoate over L-lactate by a factor of 40 (kcat/Km)
L65A
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reaction proceeds slower than in wild type
L65A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
N69K
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reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
N69K
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P44A
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reaction proceeds slower than in wild type
P44A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P64Q
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less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
P64Q
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P64R
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less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
P64R
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
V70M
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less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochromb2
V70M
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
Y143F
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Y143F
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turnover reduced to 15%
Y254F
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Y254F
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significantly reduced activity of mutant enzyme
Y254F
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only traces of activity
Y254F
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increase in Km-value, 4fold decrease of vmax
Y254F
the mutant enzyme has a Vmax value some 28fold lower than that of the wild type enzyme and a slightly raised L-lactate Km value
Y254L
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turnover reduced to less than 10%
Y254L
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only traces of activity
Y254L
site-directed mutagenesis, while the wild-type mutant has residue R289 in a distal or a proximal conformation, the mutant shows R289 only in a distal conformation
Y74F
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activity and inhibitory profile similar to wild type
Y74F
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
additional information
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enzyme knockout by construction of three T-DNA insertion lines for GOX3, gox3-1 (GK_523D09), gox3-2 (SALK_020909), and gox3-3 (SAIL_1156_F03), with insertions in exon 10, intron 10, and the 5' untranslated region, respectively. Neither the GOX3 loss-of-function mutants nor the overexpression lines showed phenotypical differences when compared with the wild type in standard growth conditions in both long- and short-day photoperiods. Loss of function of GOX3 induces metabolic rearrangements in roots that mirror wild-type responses under hypoxia. Conditional phenotype of gox3 mutant plants and complementation, overview
additional information
construction of a lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells. Combination of recombinant technology and nanotechnology by recombinant overexpression of the enzyme in Hansenula polymorpha yeast cells additionally enriched by recombinant enzyme bound with gold nanoparticles, FC b2-nAu. The FC b2-nAu-enriched livingand permeabilized yeast cells are used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulfate serves as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. The output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5fold higher compared to control cells. For intact cells, the treatment with FC b2-nAu results in 1.56fold increased enzymatic activity. For permeabilized cells, the enrichment efficacy is 2.33fold increased. Method evaluation
additional information
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construction of a lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells. Combination of recombinant technology and nanotechnology by recombinant overexpression of the enzyme in Hansenula polymorpha yeast cells additionally enriched by recombinant enzyme bound with gold nanoparticles, FC b2-nAu. The FC b2-nAu-enriched livingand permeabilized yeast cells are used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulfate serves as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. The output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5fold higher compared to control cells. For intact cells, the treatment with FC b2-nAu results in 1.56fold increased enzymatic activity. For permeabilized cells, the enrichment efficacy is 2.33fold increased. Method evaluation
additional information
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three different deletion mutants with deletion of 3, 6 and 9 amino acids from hinge region
additional information
the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow. This is not unexpected in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core. Reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c
additional information
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the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow. This is not unexpected in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core. Reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c
additional information
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for detection of the enzyme activity, recombinant enzyme is immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiratio, overview
additional information
introduction of a number of heme domain side chain substitutions in and around the interface to probe their effect on flavin to heme and cytochrome b2 electron transfer, overview
additional information
active site structures of wild-type and mutant enzymes, detailed overview
additional information
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enzyme-deficient mutant cells can be complementated by expression of GOX3, a glycolate oxidase from Arabdiospsis thaliana, which is capable to synthesize L-lactate from pyruvate, overview