Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2

Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Microbiological Research (Impact Factor: 2.56). 02/2006; 161(2):138-44. DOI: 10.1016/j.micres.2005.07.004
Source: PubMed


Gentisate 1,2-dioxygenase (GDO, EC 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.

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    • "Experimentally directed mutagenesis methods include: insertional mutagenesis [14], PCR mutagenesis [15] [16], signature tagged mutagenesis [17], site-directed mutagenesis [18] [19] and transposon mutagenesis [20] [21] [22]. Different procedures are involved in traditional mutagenesis, including molecular cloning that depends on preparations of vector and inserted DNA fragments and ligations. "
    Applied Biological Engineering - Principles and Practice, 03/2012; , ISBN: 978-953-51-0412-4
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    • "All rights reserved. doi:10.1016/j.micres.2008.08.001 number: AY648560), and four of them (mhbDHIM) were demonstrated to encode the enzymes involved in the catabolism of 3HBA to fumarate and pyruvate via the gentisate pathway (Gao, 2003; Liu et al., 2005a; Luo et al., 2006). mhbR was divergently transcribed from the 3HBA catabolic genes and encodes a 37-kDa polypeptide, which is homologous to members of the LysR family, the largest family of prokaryotic regulatory proteins identified so far (Henikoff et al., 1988). "
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    ABSTRACT: In Klebsiella pneumoniae M5a1, mhbTDHIM genes are involved in 3-hydroxybenzoate catabolism via the gentisate pathway. mhbR, which encodes a LysR-type transcriptional regulator, is divergently transcribed from the mhb structural genes. MhbR was found to be necessary for the expression of catabolic genes. Transcriptional studies demonstrated that the mhb structural genes are transcribed as an operon. The promoters of mhbR and the mhb operon are sigma(70)-type and overlap with each other. 5' Deletion analysis of the promoter transcription activity showed that a 233bp fragment (position -144 to +89 according to the transcriptional start site of mhb operon) contained the element necessary for induction. beta-Galactosidase activity assays and electrophoretic mobility shift assays showed that an inverted repeat sequence site 1 (ATAACCTCCAGGTTAT, position -70 to -55) within this fragment was critical for regulation.
    Microbiological Research 10/2008; 165(1):66-74. DOI:10.1016/j.micres.2008.08.001 · 2.56 Impact Factor
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    ABSTRACT: Salicylate 1,2-dioxygenase, a new ring-fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring-fission mechanism, has been crystallized. Diffraction-quality crystals of salicylate 1,2-dioxygenase were obtained using the sitting-drop vapour-diffusion method at 277 K from a solution containing 10%(w/v) ethanol, 6%(w/v) PEG 400, 0.1 M sodium acetate pH 4.6. Crystals belong to the primitive tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = 133.3, c = 191.51 A. A complete data set at 100 K extending to a maximum resolution of 2.9 A was collected at a wavelength of 0.8423 A. Molecular replacement using the coordinates of known extradiol dioxygenases structures as a model has so far failed to provide a solution for salicylate 1,2-dioxygenase. Attempts are currently being made to solve the structure of the enzyme by MAD experiments using the anomalous signal of the catalytic Fe(II) ions. The salicylate 1,2-dioxygenase structural model will assist in the elucidation of the catalytic mechanism of this ring-fission dioxygenase from P. salicylatoxidans, which differs markedly from the known gentisate 1,2-dioxygenases or 1-hydroxy-2-naphthoate dioxygenases because of its unique ability to oxidatively cleave salicylate, gentisate and 1-hydroxy-2-naphthoate with high catalytic efficiency.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2006; 62(Pt 6):553-5. DOI:10.1107/S1744309106016435 · 0.53 Impact Factor
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