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R190A
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the catalytic constant, kcat, of the mutant in the presence of NADH decreases 10fold while the Km for NADH decreases 12fold. The mutant shows no activity with NADPH
R82A
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the mutation leads to a 10fold increase in the Km for NADPH but does not affect the kinetics of NADH
R82D
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the mutation leads to a 10fold increase in the Km for NADPH but does not affect the kinetics of NADH
S195A
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the mutation has no effect on the affinity of the enzyme for NADPH and its affinity for NADH and for BPGA in the presence of NADH is reduced
A(plusCTE)
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chimeric mutant for testing the regulatory function of CTE
B(E326Q)
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site specific mutant of the GAPDH B-subunit
B(minCTE)
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deletion mutant for testing the regulatory function of CTE
B(R77A)
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site specific mutant of the GAPDH B-subunit
B(S188A)
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site specific mutant of the GAPDH B-subunit
C302A
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compared to wild-type enzyme the amount of the thermolabile species is higher
E268Q
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attacking water molecule in the hydrolysis process is poorly activated, can be overcome by the nulceophiles hydrazin and hydroxylamine
N169T
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compared to wild-type enzyme the amount of the thermolabile species is significantly lower
R124L
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turnover number is 21fold lower than turnover-number of wild-type enzym
R301L
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turnover number is 1000fold lower than turnover-number of wild-type enzyme. Rate limiting step is acylation, compared to deacylation in wild-type enzyme
T195G
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compared to wild-type enzyme the amount of the thermolabile species is similar
Y170F
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turnover number is 1.7fold higher than turnover-number of wild-type enzyme
E141D
kcat for D-glyceraldehyde 3-phosphate is 2.1fold lower than wild-type value
K137E
kcat for D-glyceraldehyde 3-phosphate is 1.1fold lower than wild-type value
L138T
kcat for D-glyceraldehyde 3-phosphate is 1.3fold lower than wild-type value
R136K
kcat for D-glyceraldehyde 3-phosphate is 2.8fold lower than wild-type value
Y139R
kcat for D-glyceraldehyde 3-phosphate is 302fold lower than wild-type value. The mutant enzyme no longer displays a sigmoidal K-type-like allostery but instead has apparent V-type allostery similar to that of the Sulfolobus solfataricus enzyme, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior
A198S/S199I
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the catalytic efficiency with NADP+ decreases while that with NAD+ increases by 2.5fold. Substitutions reduces the NADP/NAD preference ratio by more than 50%
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E141D
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kcat for D-glyceraldehyde 3-phosphate is 2.1fold lower than wild-type value
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K137E
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kcat for D-glyceraldehyde 3-phosphate is 1.1fold lower than wild-type value
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L138T
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kcat for D-glyceraldehyde 3-phosphate is 1.3fold lower than wild-type value
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R136K
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kcat for D-glyceraldehyde 3-phosphate is 2.8fold lower than wild-type value
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S199I
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the catalytic efficiency (kcat/Km) with NADP+ decreases by 0.5fold while that with NAD+ remains unchanged. Substitution reduces the NADP/NAD preference ratio by more than 50%
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E268A
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attacking water molecule in the hydrolysis process is poorly activated, can be overcome by the nulceophiles hydrazin and hydroxylamine
E268A
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compared to wild-type enzyme the amount of the thermolabile species is significantly lower
E268A
mutant shows reduced deacylation rate
R459I
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compared to wild-type enzyme the amount of the thermolabile species is significantly lower
R459I
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turnover number is 214fold lower than turnover-number of wild-type enzyme. Rate limiting step is acylation, compared to deacylation in wild-type enzyme
A198S/S199I
the catalytic efficiency with NADP+ decreases while that with NAD+ increases by 2.5fold. Substitutions reduces the NADP/NAD preference ratio by more than 50%
A198S/S199I
the mutant shows reduced catalytic efficiency with NADP+ and increased catalytic efficiency with NAD+ as compared to the wild type enzyme
S199I
the catalytic efficiency (kcat/Km) with NADP+ decreases by 0.5fold while that with NAD+ remains unchanged. Substitution reduces the NADP/NAD preference ratio by more than 50%
S199I
the mutant shows reduced catalytic efficiency with NADP+ and increased catalytic efficiency with NAD+ as compared to the wild type enzyme
additional information
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replacement of Escherichia coli GapA glyceraldehyde 3-phosphate dehydrogenase, EC 1.2.1.12 by Clostridium acetobutylicum GapC glyceraldehyde 3-phosphate dehydrogenase, EC 1.2.1.9 results in significant reduction of flux through the pentose phosphate pathway. Recombinant strains display increased NADPH availability, and consistently higher productivity than parent strains
additional information
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CRISPR-Cpf1-assisted engineering of Corynebacterium glutamicum SNK118 for enhanced L-ornithine production by NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and NADH-dependent glutamate dehydrogenase. Insufficient intracellular NADPH content has been recognized as a limiting factor in L-ornithine biosynthesis. Overexpression of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase gene CsgapC from Clostridium saccharobutylicum strain DSM 13864 and NADH-dependent glutamate dehydrogenase gene BsrocG from Bacillus subtilis strain HB-1 results in markedly increased ornithine biosynthesis. The engineered Corynebacterium glutamicum strain KBJ11 (SNK118DELTAargRDELTAargFDELTAncgl2228/pXMJ19-CsgapC-BsrocG) is constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/l with productivity of 1.23 g/l/h (over 72 h) and yield of 0.414 g/g glucose is achieved by strain KBJ11 in a 10-l bioreactor. High titer and yield of L-ornithine production by microbial fermentation proves that heterologous expression of CsgapC and BsrocG can promote L-ornithine production by Corynebacterium glutamicum strains
additional information
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CRISPR-Cpf1-assisted engineering of Corynebacterium glutamicum SNK118 for enhanced L-ornithine production by NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and NADH-dependent glutamate dehydrogenase. Insufficient intracellular NADPH content has been recognized as a limiting factor in L-ornithine biosynthesis. Overexpression of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase gene CsgapC from Clostridium saccharobutylicum strain DSM 13864 and NADH-dependent glutamate dehydrogenase gene BsrocG from Bacillus subtilis strain HB-1 results in markedly increased ornithine biosynthesis. The engineered Corynebacterium glutamicum strain KBJ11 (SNK118DELTAargRDELTAargFDELTAncgl2228/pXMJ19-CsgapC-BsrocG) is constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/l with productivity of 1.23 g/l/h (over 72 h) and yield of 0.414 g/g glucose is achieved by strain KBJ11 in a 10-l bioreactor. High titer and yield of L-ornithine production by microbial fermentation proves that heterologous expression of CsgapC and BsrocG can promote L-ornithine production by Corynebacterium glutamicum strains
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additional information
in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
additional information
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in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
additional information
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in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
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additional information
the gapA gene, UniProt ID P0A9B2, EC 1.2.1.12, from Escherichia coli strain MG1655 is replaced by the gene gapN from Streptococcus mutans. The specific NADP+-GAPDH activity of the strain MG1655DgapA::gapN is 4.6times lower relative to strain MG1655DELTAgapA::gapN/pTrcgapN and no NAD+-GAPDH activity is detected. The specific NADP+-GAPDH activity levels in the derivative strain reveal that growth rate and glucose uptake differences are attributable to gapN expression level. The NADH/NAD+ ratio in the strain MG1655DELTAgapA::gapN/pTrcgapN decreases by 25% as compared to wild-type strain. In contrast, the NADPH/NADP+ ratio increases 2times indicating that the alteration in the turnover of NAD(P)H via glyceraldehyde 3-phosphate oxidation affects the redox levels of the strain MG1655DELTAgapA::gapN/pTrcgapN, which increases 2.8times the NADPH/NADH ratio
additional information
in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
additional information
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in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
additional information
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in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii, D-glucose 1-phosphate-induced activation follows an increase in affinity for glyceraldehyde-3-phosphate rather than an increase in maximum velocity, whereas in the enzyme from Sulfolobus solfataricus, D-glucose 1-phosphate-induced activation follows an increase in maximum velocity rather than in affinity for glyceraldehyde-3-phosphate. To explore the molecular basis of this difference 14 mutants and 2 chimeras are generated. The analyses of chimeric enzymes generated from regions of the Sulfolobus tokodaii enzyme and the Sulfolobus solfataricus enzyme indicates that a 57-residue module located in the subunit interface is involved in their allosteric behavior
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