1.13.11.4 A112D the mutant gains the ability to oxidize salicylate and several other monohydroxylated substrates -, 743728 1.13.11.4 A112G mutation does not alter the nature of the Fe-substrate-O2 ternary complex 765091 1.13.11.4 A112G the mutant gains the ability to oxidize salicylate and several other monohydroxylated substrates -, 743728 1.13.11.4 A112I the mutant gains the ability to oxidize salicylate and several other monohydroxylated substrates -, 743728 1.13.11.4 A112S the mutant gains the ability to oxidize salicylate and several other monohydroxylated substrates -, 743728 1.13.11.4 D120K complete loss of activity -, 674253 1.13.11.4 D175N inactive mutant enzyme 689914 1.13.11.4 D225N activity is comparable to wild-type activity 689914 1.13.11.4 E223A 102% relative activity compared to the wild type enzyme 674253 1.13.11.4 E47Q activity is comparable to wild-type activity 689914 1.13.11.4 F159Y site-directed mutagenesis 724975 1.13.11.4 G106A site-directed mutagenesis, in contrast to the wild-type enzyme, mutant G106A oxidizes only gentisate, while 1-hydroxy-2-naphthoate and salicylate are not converted. The mutant shows slightly decreased activity with salicylate, but shows a higher affinity to this substratecompared to the wild-type. Salicylate is coordinated in the G106A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that is formed between Trp104 and Asp174 in the G106A variant -, 724975 1.13.11.4 G111A site-directed mutagenesis -, 724975 1.13.11.4 G123N 75% relative activity compared to the wild type enzyme -, 674253 1.13.11.4 G164T complete loss of activity 674253 1.13.11.4 H108D complete loss of activity -, 675818 1.13.11.4 H108D no enzymatic activity 389991 1.13.11.4 H110D complete loss of activity -, 675818 1.13.11.4 H110D no enzymatic activity 389991 1.13.11.4 H118D complete loss of activity 675818 1.13.11.4 H119A/H121A despite its wild-type like structural propertie, the mutant shows extremely low gentisate 1,2-dioxygenase activity 689914 1.13.11.4 H120D complete loss of activity 675818 1.13.11.4 H149D complete loss of activity -, 675818 1.13.11.4 H149D no enzymatic activity 389991 1.13.11.4 H151D complete loss of activity -, 675818 1.13.11.4 H151D no enzymatic activity 389991 1.13.11.4 H155A inactive 743798 1.13.11.4 H157A inactive 743798 1.13.11.4 H159D complete loss of activity 675818 1.13.11.4 H161D complete loss of activity -, 675818 1.13.11.4 H167A inactive 743798 1.13.11.4 H169A inactive 743798 1.13.11.4 H290A/H292A mutant can not be expressed in a soluble form 689914 1.13.11.4 K338Y 156% relative activity compared to the wild type enzyme 674253 1.13.11.4 L39T activity is comparable to wild-type activity 689914 1.13.11.4 M103L site-directed mutagenesis -, 724975 1.13.11.4 M146T complete loss of activity 674253 1.13.11.4 M169T complete loss of activity 674253 1.13.11.4 additional information construction of individual knockout mutants of all three nagI genes via 2 bp unmarked gene deletion mutagenesis, all three nagI knockout mutants still contain stop codons within their nagI genes. Mutant CJ2DELTAnagI3 shows severely diminished GDO activity and grows slowest on aromatic substrates 725905 1.13.11.4 additional information double mutant Phe3Leu, Val334Ala, doubled enzymatic activity, mutants in conserved core regions with less activity 389991 1.13.11.4 additional information knockout mutant G56 -, 658819 1.13.11.4 N153H 183% relative activity compared to the wild type enzyme 674253 1.13.11.4 N43T 317% relative activity compared to the wild type enzyme -, 674253 1.13.11.4 P253_Y254del mutation reduces the activity to 30% 689914 1.13.11.4 Q108E inactive mutant enzyme 689914 1.13.11.4 Q108E/D175N activity is near zero 689914 1.13.11.4 R113G site-directed mutagenesis -, 724975 1.13.11.4 S113P complete loss of activity 674253 1.13.11.4 S147R site-directed mutagenesis -, 724975 1.13.11.4 T260C complete loss of activity 674253 1.13.11.4 V326Q complete loss of activity 674253 1.13.11.4 V36A complete loss of activity 674253 1.13.11.4 Y17C 161% relative activity compared to the wild type enzyme -, 674253 1.13.11.4 Y181D 68% relative activity compared to the wild type enzyme 674253 1.13.11.4 Y181F mutant demonstrates 4-, 3-, 6-, and 16fold increase in relative activity towards 2,5-dihydroxybenzoate, 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively, and shows 464% relative activity compared to the wild type enzyme for 2,5-dihydroxybenzoate -, 674253 1.13.11.4 Y181H 98% relative activity compared to the wild type enzyme 674253 1.13.11.4 Y284I 260% relative activity compared to the wild type enzyme 674253