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: 1.94). 02/2006; 161(2):138-44. DOI: 10.1016/j.micres.2005.07.004
Source: PubMed

ABSTRACT 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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    Applied Biological Engineering - Principles and Practice, 03/2012; , ISBN: 978-953-51-0412-4
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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. · 1.94 Impact Factor
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    ABSTRACT: Klebsiella pneumoniae M5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene cluster mhbRTDHIM conferred the ability to grow on 3-hydroxybenzoate to Escherichia coli and Pseudomonas putida PaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H(+) symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane. P. putida PaW340 containing mhbRΔTDHIM could not grow on 3-hydroxybenzoate; however, supplying mhbT in trans allowed the bacterium to grow on the substrate. K. pneumoniae M5a1 and P. putida PaW340 containing recombinant MhbT transported (14)C-labeled 3-hydroxybenzoate but not (14)C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake by P. putida PaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.
    Applied and Environmental Microbiology 06/2012; 78(17):6113-20. · 3.95 Impact Factor


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