1.2.1.79 C262A active site mutation, nonfunctional because Cys-262 acts as a nucleophilel 725523 1.2.1.79 C262A mutation abolishes catalytic activity, catalytic residue 725838 1.2.1.79 C262A site-directed mutagenesis, Sp2771 mutant structure analysis and comparison to the wild-type structure 743181 1.2.1.79 C289A site-directed mutagenesis, inactive mutant 763015 1.2.1.79 C291A site-directed mutagenesis, the mutant shows 65% activity compared to wild-type enzyme 763015 1.2.1.79 E228A active site mutation, nonfunctional because Glu-228 acts as a general base 725523 1.2.1.79 E228A mutation abolishes catalytic activity, catalytic residue 725838 1.2.1.79 E228A site-directed mutagenesis, inactive mutant 763137 1.2.1.79 E228D sitedirected mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme 763137 1.2.1.79 E228Q active site mutation, nonfunctional because Glu-228 acts as a general base 725523 1.2.1.79 F132A activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity 725523 1.2.1.79 F425A inactive, suggesting that Phe-425 plays an important role in substrate binding 725523 1.2.1.79 I263A activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity 725523 1.2.1.79 additional information metabolic engineering of Corynebacterium glutamicum for the production of glutaric acid, a C5 dicarboxylic acid platform chemical, by co-expression of Pseudomonas putida davT, davB, and davD genes encoding lysine 2-monooxygenase, delta-aminovaleramidase, and glutarate semialdehyde dehydrogenase, respectively, in Corynebacterium glutamicum. Method optimization and evaluation. The glutaric acid biosynthesis pathway constructed in recombinant Corynebacterium glutamicum is engineered by examining strong synthetic promoters H30 and H36, Corynebacterium glutamicum codon-optimized davTDBA genes, and modification of davB gene with an N-terminal His6-tag to improve the production of glutaric acid. The use of N-terminal His6-tagged DavB is most suitable for the production of glutaric acid from glucose. Fed-batch fermentation of the final engineered Corynebacterium glutamicum H30_GAHis strain, expressing davTDA genes along with davB fused with His6-tag at N-terminus can produce 24.5 g/l of glutaric acid with low accumulation of L-lysine (1.7 g/l), wherein 5-aminovaleric acid (5-AVA) ccumulation is not observed during fermentation. Metabolically engineered Corynebacterium glutamicum strain KCTC H30_GA-2 (engineered strain KCTC 1857) is able for catalysis of the biosynthesis of glutaric acid from glucose. Method optimization and evaluation, overview 754623 1.2.1.79 N131A mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity 725523 1.2.1.79 N131A site-directed mutagenesis, inactive mutant 763137 1.2.1.79 N131D mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity 725523 1.2.1.79 N131D site-directed mutagenesis, inactive mutant 763137 1.2.1.79 N175A site-directed mutagenesis, the mutant shows reduced activity compared tow wild-type enzyme 763578 1.2.1.79 R121A site-directed mutagenesis, almost inactive mutant 763578 1.2.1.79 R139A 90% reduced catalytic activity, residue is involved in substrate binding 725838 1.2.1.79 R139A activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity 725523 1.2.1.79 R139A site-directed mutagenesis, the mutant displays catalytic efficiency (kcat/Km) of only respective 0.2% compared to wild-type enzyme with significantly decreased binding affinity for succinic semialdehyde 763137 1.2.1.79 R139K mutant enzyme exhibited an activity up to 80% that of the wild type enzyme, suggesting the significance of a positively charged residue in the binding of the carboxyl group of succinic semialdehyde 725523 1.2.1.79 R228A site-directed mutagenesis, the mutation results in 37fold lower catalytic efficiency value (kcat/Km) for NADP+, but only fourfold lower value for NAD+ compared to wild-type 763578 1.2.1.79 R457A site-directed mutagenesis, almost inactive mutant 763578 1.2.1.79 S157E mutation changes cofactor preference from NADP+ to NAD+, but enzyme activity is approximately 10fold reduced 725838 1.2.1.79 S157E site-directed mutagenesis, the mutant shows altered cofactor specificity compared to wild-type, preferring NAD+, mutation of Ser157 does not significantly affect the binding affinity of SSA with the enzyme 763137 1.2.1.79 S157P site-directed mutagenesis, the mutant shows altered cofactor specificity compared to wild-type, preferring NAD+, mutation of Ser157 does not significantly affect the binding affinity of SSA with the enzyme 763137 1.2.1.79 S419A 80% reduced catalytic activity, residue is involved in substrate binding 725838 1.2.1.79 S419A mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity 725523 1.2.1.79 S419A site-directed mutagenesis, Sp2771 mutant structure analysis and comparison to the wild-type structure 743181 1.2.1.79 S420A site-directed mutagenesis, the mutant displays catalytic efficiency (kcat/Km) of only respective 0.4% compared to wild-type enzyme with significantly decreased binding affinity for succinic semialdehyde 763137 1.2.1.79 W135A activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity 725523 1.2.1.79 Y296A site-directed mutagenesis, the mutant shows reduced activity compared tow wild-type enzyme 763578