Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2.
Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is the first enzyme in gentisate pathway that catalyses the ring fission of gentisate to form maleylpyruvate. Phylogenetic tree of amino acid sequences from 11 GDOs demonstrates that the GDOs from different genus share identities between 12.1% and 64.8%. According to the alignment result, four highly conserved histidine residues in GDO from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2 were chosen to be substituted with aspartate residues. Enzyme analysis indicated that substitution of any of these four histidine residues had resulted in the complete loss of its catalytic activity.
Luo
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Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Liu
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Liu
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Zhou
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D043367:Ralstonia / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D006639:Histidine; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase