Molecular and Biochemical Characterization of the
            <i>xlnD</i>
            -Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in
            <i>Pseudomonas alcaligenes</i>
            NCIMB 9867

Gao, Xiaoli; Tan, Chew Ling; Yeo, Chew Chieng; Poh, Chit Laa

doi:10.1128/jb.187.22.7696-7702.2005

Cited by 36 publications

(25 citation statements)

References 31 publications

(43 reference statements)

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“…Presently, the sequences of only two genes known to encode 3-hydroxybenzoate 6-hydroxylase are available: those of mhbM from K. pneumoniae M5a1 (18) and xlnD from P. alcaligenes NCIMB 9867 (6). The NagX enzyme from strain CJ2 was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, two related proteins (MhbM from Klebsiella pneumoniae M5a1 [18] and XlnD from Pseudomonas alcaligenes NCIMB 9867 [6]) with low levels of amino acid sequence identity to Orf2 (55.6 and 72.2%, respectively) have been reported to act as 3-hydroxybenzoate 6-hydroxylases. Additional information available on the genetics and biochemistry of 3-hydroxybenzoate 6-hydroxylase is limited largely to that from studies using five model organisms, K. pneumoniae (13,18,30), P. alcaligenes (6,21,22), Burkholderia (formerly Pseudomonas) cepacia (23), Micrococcus sp. (33), and Pseudomonas aeruginosa (8).…”

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confidence: 99%

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Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Park¹,

Jeon²,

Jang³

et al. 2007

Appl Environ Microbiol

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Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI-orf1-tnpA and nagR2-orf2I؆KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.Polaromonas naphthalenivorans CJ2, previously found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (10), is able to utilize naphthalene as the sole carbon source. Recent sequencing and analysis of the entire naphthalene degradation pathway of P. naphthalenivorans CJ2 (12) revealed that the naphthalene-catabolic (nag) genes in P. naphthalenivorans CJ2 are homologous to those in Ralstonia sp. strain U2, which metabolizes naphthalene via the gentisate pathway, converting naphthalene into fumarate and pyruvate via salicylate (2-hydroxybenzoate) and gentisate (2,5-dihydroxybenzoate) (5). This finding has recently been confirmed biochemically (G. M. Pumphrey and E. L. Madsen, submitted for publication). In contrast to the nag genes of strain U2, which are organized in a single operon, the naphthalenecatabolic genes of strain CJ2 are organized into two gene clusters (Fig. 1A), comprising nagRAaGHAbAcAdBFCQEDJIЈ-orf1-tnpA (large cluster) and nagR2-orf2IЉKL (small cluster). LysR-type (nagR) and MarR-type (nagR2) transcriptional regulators act, respectively, to up-regulate and down-regulate the growth of strain CJ2 on naphthalene in min...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Park¹,

Jeon²,

Jang³

et al. 2007

Appl Environ Microbiol

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“…A set of genes might encode catabolic pathway for aerobic metabolism of 3-hydroxybenzoate (3-HBA) is predicted in strain S. pomeroyi DSS-3 based on the previous works (Jones and Cooper, 1990;Zhou et al, 2001;Gao et al, 2005;Shen et al, 2005) (Table 4). It's been known that there are three downstream routes for the further catalysis of maleylpyruvate, the product from gentisate 1, 2-dioxygenase, namely, maleylpyruvate is either cleaved directly to form pyruvate and maleate by the enzyme maleylpyruvate hydrolase or converted to fumarylpyruvate with maleylpyruvate isomerase glutathione-independent (Crawford and Frick, 1977;Shen et al, 2005) or glutathione-dependent (Lack, 1959(Lack, , 1961Crawford and Frick, 1977;Ishiyama, 2004).…”

Section: The Gentisate Pathwaymentioning

confidence: 99%

Genomic analysis of the aromatic catabolic pathways fromSilicibacter pomeroyi DSS-3

2009

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Genomic analysis of the catabolic potentialities of Silicibacter pomeroyi DSS-3 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least seven main pathways for the conversion of the aromatic compounds to the intermediates which enter into TCA cycle, that is, the catechol (cat I and cat II genes) and protocatechuate (pca genes) branches of the β-ketoadipate pathway, the phenylacetate pathway (paa genes), the gentisate pathway (gtd genes), the homogentisate pathway (hmg/hppD genes), as well as the homoprotocatechuate pathway (hpc genes). Furthermore, the genes encoding those enzymes involved in the peripheral pathways leading to the β-ketoadipate central pathway were also mapped, i.e., 4-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and act), tyrosine (hpp) and n-phenylalkanoic acids (fad). Evidences showed that S. pomeroyi DSS-3 have versatile abilities to the catabolism of aromatic compounds either in anaerobic or in aerobic pathway, suggesting such a strain might be a model of heuristic value for the study of the genomic organization, the evolution of genes, as well as the catalytic or transcriptional mechanisms of enzymes for aromatic degradation in marine bacteria. Further, it would provide new insights into the biodegradation of aromatic compounds in marine bacteria and marine environments.

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“…strain NCIMB 12038 (28), Polaromonas naphthalenivorans CJ2 (35), and Pseudomonas alcaligenes NCIMB 9867 (12), but no transporter for 3-hydroxybenzoate uptake has been reported so far. Notably, when 3-hydroxybenzoate catabolism in the Gram-positive organism Corynebacterium glutamicum was studied, a putative transporter gene within the catabolic cluster turned out to be involved in gentisate catabolism rather than 3-hydroxybenzoate catabolism (40).…”

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MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Gao

Wang

et al. 2012

Appl Environ Microbiol

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ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. 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. putidaPaW340 containingmhbRΔTDHIMcould not grow on 3-hydroxybenzoate; however, supplyingmhbTintransallowed the bacterium to grow on the substrate.K. pneumoniaeM5a1 andP. putidaPaW340 containing recombinant MhbT transported14C-labeled 3-hydroxybenzoate but not14C-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 byP. putidaPaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.

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Molecular and Biochemical Characterization of the xlnD -Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

Cited by 36 publications

References 31 publications

Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Genomic analysis of the aromatic catabolic pathways fromSilicibacter pomeroyi DSS-3

MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

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