2.7.2.8 A26V site-directed mutagenesis, the mutation deregulates the feedback inhibition of the enzyme 738801 2.7.2.8 D162E about 0.1% of wild-type activity 659704 2.7.2.8 DELTA2-15 Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 71fold increased compared to wild-type 722087 2.7.2.8 DELTA2-19 Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 148fold increased compared to wild-type 722087 2.7.2.8 DELTA2-29 Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 163fold increased compared to wild-type 722087 2.7.2.8 DELTA25 Km (N-acetyl-L-glutamate) highly increased compared to wild-type, kcat highly decreased compared to wild-type, IC50 (L-arginine) 17fold increased compared to wild-type 722087 2.7.2.8 DELTA357-513 truncated mutant lacking C-terminal 150 amino acids (spanning residues 38-356), belonging to the DUF619 domain family, shows that is it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. Truncated yNAGK shows doubled kcat compared to wild-type, Km is almost not affected. IC50 (L-arginine) is lowered compared to wild-type. Truncated mutand shows lower thermal stability compared to wild-type 723557 2.7.2.8 DELTA8 Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat highly decreased compared to wild-type, IC50 (L-arginine) 16fold increased compared to wild-type 722087 2.7.2.8 E17A site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine 692856 2.7.2.8 E17D site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine 692856 2.7.2.8 E17Q site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine 692856 2.7.2.8 E186A/E387A to improve resolution in crystallization 671144 2.7.2.8 E19A Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 40fold increased compared to wild-type 722087 2.7.2.8 E19R Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 61fold increased compared to wild-type 722087 2.7.2.8 E19R Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type 721268 2.7.2.8 E19R site-directed mutagenesis, the mutation increases the 50% inhibitoryL--arginine concentration significantly in vitro, which deregulates the feedback inhibition of CcNAGK by L-arginine in SYPA5-5 during the fermentation 738801 2.7.2.8 E281A Km (N-acetyl-L-glutamate) highly increased compared to wild-type, kcat similar to wild-type, IC50 (L-arginine) 21fold increased compared to wild-type 722087 2.7.2.8 E284D site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 E416A/K417A to improve resolution in crystallization 671144 2.7.2.8 E94A/K95A to improve resolution in crystallization 671144 2.7.2.8 G11A G11A does not hamper recombinant enzyme expression or purification. Mutant shows a 10fold decrease in Vmax values and it selectively increases 8fold the Km for ATP, affecting much less (3fold increase) the apparent Km for N-acetyl-L-glutamate 722965 2.7.2.8 G287A Km (N-acetyl-L-glutamate) similar to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 37fold increased compared to wild-type 722087 2.7.2.8 G287D Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 40fold increased compared to wild-type 722087 2.7.2.8 G287D site-directed mutagenesis, the mutation increases the 50% inhibitoryL--arginine concentration significantly in vitro, which deregulates the feedback inhibition of CcNAGK by L-arginine in SYPA5-5 during the fermentation 738801 2.7.2.8 G290A site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 H268A Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 39fold increased compared to wild-type 722087 2.7.2.8 H268N Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 58fold increased compared to wild-type 722087 2.7.2.8 H268N Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type 721268 2.7.2.8 H268N site-directed mutagenesis, the mutant strain shows accumulation of large amounts of pathway intermediates L-citrulline and L-ornithine and decreased production of L-arginine. Transcription levels of argGH decrease accompanied with the reduction of argininosuccinate synthase activity, which leads to the metabolic obstacle from L-citrulline to L-arginine -, 738801 2.7.2.8 H268N site-directed mutagenesis, the mutation increases the 50% inhibitoryL--arginine concentration significantly in vitro, which deregulates the feedback inhibition of CcNAGK by L-arginine in SYPA5-5 during the fermentation -, 738801 2.7.2.8 H268N the mutation significantly decreases the sensitivity of the enzyme to L-arginine and keeps the specific activity as compared to the wild type enzyme -, 761643 2.7.2.8 H268N/H26E Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) 875fold increased compared to wild-type 721268 2.7.2.8 H268N/H26E/E19R Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) 1960fold increased compared to wild-type 721268 2.7.2.8 H26A Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 54fold increased compared to wild-type 722087 2.7.2.8 H26E Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type 721268 2.7.2.8 H26E site-directed mutagenesis, the mutation increases the 50% inhibitoryL--arginine concentration significantly in vitro, which deregulates the feedback inhibition of CcNAGK by L-arginine in SYPA5-5 during the fermentation 738801 2.7.2.8 H271N site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 I106M to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A 720845 2.7.2.8 I294M to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A 720845 2.7.2.8 K213 site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 K26A/E27A to improve resolution in crystallization 671144 2.7.2.8 K279A/E280A to improve resolution in crystallization 671144 2.7.2.8 K356H site-directed mutagenesis, inactive mutant 735596 2.7.2.8 K364H site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme -, 735596 2.7.2.8 K419A to improve resolution in crystallization 671144 2.7.2.8 K8R substantial although diminished activity 659704 2.7.2.8 L367M to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A 720845 2.7.2.8 M31V site-directed mutagenesis, the mutation deregulates the feedback inhibition of the enzyme 738801 2.7.2.8 additional information construction of feedback inhibition deregulated Corynebacterium crenatum mutant strains, phenotypes, overview -, 738801 2.7.2.8 additional information N-helix N-terminal deletions spanning 16 residues dissociate NAGK to active dimers, those of 20 residues decrease the apparent affinity for arginine, and complete N-helix deletion of 26 residues abolishes arginine inhibition, overview 692856 2.7.2.8 N158K substantial although diminished activity 659704 2.7.2.8 N391Q site-directed mutagenesis, inactive mutant 735596 2.7.2.8 N399Q site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme -, 735596 2.7.2.8 Q10A site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 R209A Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 46fold increased compared to wild-type 722087 2.7.2.8 R209A site-directed mutagenesis, the mutant strain shows accumulation of large amounts of pathway intermediates L-citrulline and L-ornithine and decreased production of L-arginine. Transcription levels of argGH decrease accompanied with the reduction of argininosuccinase activity, which leads to the metabolic obstacle from L-citrulline to L-arginine -, 738801 2.7.2.8 R209A site-directed mutagenesis, the mutation increases the 50% inhibitoryL--arginine concentration significantly in vitro, which deregulates the feedback inhibition of CcNAGK by L-arginine in SYPA5-5 during the fermentation -, 738801 2.7.2.8 R209A the mutation significantly decreases the sensitivity of the enzyme to L-arginine and keeps the specific activity as compared to the wild type enzyme -, 761643 2.7.2.8 R209K Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 37fold increased compared to wild-type 722087 2.7.2.8 R233A mutant does not interact in vitro with PII protein of wild-type sequence in yeast two-hybrid analysis. Mutant interacts with PII variants containing I86N or I86T mutation 722963 2.7.2.8 R24E site-directed mutagenesis, the mutant shows reduced Vmax , the mutation results in increased affinity of NAGK for arginine 692856 2.7.2.8 R386K site-directed mutagenesis, inactive mutant 735596 2.7.2.8 R394K site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme -, 735596 2.7.2.8 R66K substantial although diminished activity 659704 2.7.2.8 S387A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme 735596 2.7.2.8 S395A site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme -, 735596 2.7.2.8 W23A Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 4.5fold increased compared to wild-type 722087 2.7.2.8 Y21A site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates 692856 2.7.2.8 Y397F site-directed mutagenesis, inactive mutant 735596 2.7.2.8 Y405F site-directed mutagenesis, inactive mutant -, 735596