Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: induction, purification, and characterization

Wang, Lee Ho; Hamzah, Riyad Y.; Yu, Yimin; Tu, Shiao‐Chun

doi:10.1021/bi00378a017

Cited by 52 publications

(41 citation statements)

References 21 publications

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“…(22). Burkholderia (formerly Pseudomonas) cepacia (28) and Klebsiella pneumoniae (12,25), from which the gene was recently cloned (16). Here, we show that xlnD from strain P25X does indeed encode for 3-hydroxybenzoate 6-hydroxylase I and present its biochemical and catalytic properties.…”

mentioning

confidence: 69%

“…The in vitro assay for 3-hydroxybenzoate 6-hydroxylase was based on the 3-hydroxybenzoate-linked oxidation of NADH measured at 340 nm (16,28). The end product in the sample cuvette was shown to be gentisate by the addition of the xlnE-encoded gentisate 1,2-dioxygenase (29) to the cuvette and observing for a shift in the max from 320 to 330 nm that corresponded to the conversion of gentisate to maleylpyruvate.…”

Section: Resultsmentioning

confidence: 99%

“…The 3-hydroxybenzoate 6-hydroxylase activity was determined by measuring the decrease in absorbance at 340 nm due to the substrate-dependent oxidation of NADH, the molar extinction coefficient of which was taken as 6,200 M Ϫ1 cm Ϫ1 (16,28). Assays were carried out in 100 mM potassium phosphate buffer (pH 7.4) at 23°C in a UV-2550 spectrophotometer (Shimadzu, Japan).…”

Section: Vol 187 2005mentioning

confidence: 99%

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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

et al. 2005

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The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

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confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Vol 187 2005mentioning

confidence: 99%

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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

et al. 2005

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“…␤-Galactosidase activity was determined in Miller units, as previously described (29,30). The 3-HBA 6-hydroxylase activity and GDO activity were determined as described previously (31,32). Protein concentration was determined according to the Bradford method (33).…”

Section: Construction Of Plasmids and Strainsmentioning

confidence: 99%

GenR, an IclR-Type Regulator, Activates and Represses the Transcription ofgenGenes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum

Chao

Zhou

2013

J Bacteriol

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bThe genes required for 3-hydroxybenzoate and gentisate catabolism in Corynebacterium glutamicum are closely clustered in three operons. GenR, an IclR-type regulator, can activate the transcription of genKH and genDFM operons in response to 3-hydroxybenzoate and gentisate, and it can repress its own expression. Footprinting analyses demonstrated that GenR bound to four sites with different affinities. Two GenR-binding sites (DFMn01 and DFMn02) were found to be located between positions ؊41 and ؊84 upstream of the ؊35 and ؊10 regions of the genDFM promoter, which was involved in positive regulation of genDFM transcription. The GenR binding site R-KHn01 (located between positions ؊47 and ؊16) overlapped the ؊35 region of the genKH promoter sequence and is involved in positive regulation of its transcription. The binding site R-KHn02, at which GenR binds to its own promoter, was found within a footprint extending from position ؊44 to ؊67. It appeared to be involved in negative regulation of the activity of the genR promoter. A consensus motif with a 5-bp imperfect palindromic sequence [ATTCC-N 7(5) -GGAAT] was identified among all four GenR binding sites and found to be necessary to GenR regulation through site-directed mutagenesis. The results reveal a new regulatory function of the IclR family in the catabolism of aromatic compounds.

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“…The fumarylpyruvate hydrolase used here was purified NagK from strain U2 (45) or BagK from strain NyZ101 in the present study. The 3-hydroxybenzoate 6-monooxygenase activity was determined by measuring the decrease in the absorbance at 340 nm due to the substrate-dependent oxidation of NADH, the molar extinction coefficient of which was taken as 6,200 M Ϫ1 cm Ϫ1 (40). GDO was assayed by measuring the increase in absorbance at 330 nm due to conversion of gentisate to maleylpyruvate; the molar extinction coefficient was taken as 13,000 M Ϫ1 cm Ϫ1 (25).…”

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confidence: 99%

Novel l -Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101

Liu

Zhou

2012

J Bacteriol

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Glutathione-and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G؉C Gram-positive strains. In the present study, a low-G؉C Gram-positive Paenibacillus sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (bagKLIX) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with L-cysteine, cysteinylglycine, or glutathione, as its cofactor. The K m values of these three thiols for BagL were 15.5, 8.4, and 552 M, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G؉C Gram-positive strains, BagL should utilize L-cysteine as its physiological cofactor in vivo. The addition of Ni 2؉ increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni 2؉ ion and were necessary for its enzyme activity. BagL is the first characterized L-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.

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Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: induction, purification, and characterization

Cited by 52 publications

References 21 publications

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

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

GenR, an IclR-Type Regulator, Activates and Represses the Transcription ofgenGenes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum

Novel l -Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101

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