Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867

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

doi:10.1016/j.resmic.2007.06.003

Cited by 14 publications

(11 citation statements)

References 26 publications

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“…Polaro_naph_2: Polaromonas naphthalenivorans CJ2-AAZ93397 (nagI´) -Jeon et al [47]. Polaro_naph_3: Polaromonas naphthalenivorans CJ2-ABM37760-(nagI3) -Lee et al [48]. Pseud_alcal_2: Pseudomonas alcaligenes NCIB 9867 (hbzE) ABD64513.1 -Yeo et al [49].…”

Section: Q1unclassified

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Eppinger¹,

Ferraroni²,

Bürger³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Section: Q1unclassified

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Eppinger¹,

Ferraroni²,

Bürger³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…AF173167) (29) and hbz (GenBank accession no. DQ394580) (19), were reported to be involved in gentisate catabolism in strain NCIMB 9867, which was identified to be an mcresol and 2,5-and 3,5-xylenol utilizer (30). Additionally, it was reported that strain NCIMB 9867 could spontaneously mutate to lose its ability to utilize m-cresol or 2,5-or 3,5-xylenol but still retain the ability to grow on 3-hydroxybenzoate and gentisate (9).…”

Section: Resultsmentioning

confidence: 99%

“…In the previously reported hbz cluster of P. alcaligenes NCIMB 9867 (19), two genes were identified to be involved in gentisate metabolism. hbzE encodes a strictly inducible gentisate 1,2-dioxygenase (19), and hbzF encodes a maleylpyruvate hydrolase which catalyzes the direct hydrolysis of maleylpyruvate without prior isomerization (10). Here, an additional open reading frame (ORF), designated hbzG, was identified between hbzH and hbzI, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

Liu

Zhou

2015

Appl Environ Microbiol

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In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure D-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF-disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a D-malate dehydrogenase gene was introduced into the hbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of D-malate from maleate by this D-malate dehydrogenase-deficient natural mutant. G entisate (2,5-dihydroxybenzoate) is an important ring cleavage intermediate present in the bacterial catabolic pathway of many aromatic compounds. In the gentisate pathway, gentisate 1,2-dioxygenase catalyzes the ring cleavage oxidation of gentisate to yield maleylpyruvate, which is then further degraded by either direct hydrolysis to maleate and pyruvate (1) or isomerization to fumarylpyruvate and subsequent hydrolysis to fumarate and pyruvate (2, 3). For the isomerization route, glutathione (GSH)-, mycothiol (MSH)-, or L-cysteine-dependent maleylpyruvate isomerases have been biochemically and genetically characterized in Gram-negative (4), high-GϩC-content Gram-positive (5, 6), or low-GϩC-content Gram-positive (7) bacterial strains, respectively. As an alternative to the isomerization route, maleylpyruvate can also be degraded via the direct hydrolysis route, which is catalyzed by maleylpyruvate hydrolase to yield maleate and pyruvate (Fig. 1). In contrast to the well-studied isomerization route, the direct hydrolysis route has been studied only in the 2,5-and 3,5-xylenol utilizer Pseudomonas alcaligenes NCIMB 9867 (8, 9), in which m-cresol, xylenols, and 3-hydroxybenzoate are degraded via gentisate. In this strain, a maleylpyruvate hydrolaseencoding gene, hbzF, was ch...

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“…Extraction of total RNA, reverse transcription (RT)-PCR, and real-time PCR were carried out as described previously (7). It was reported that the hbzEencoded GDO was strictly inducible by 3-hydroxybenzoate, gentisate, or 2,5-xylenol (9,12). Since hbzF was located upstream of hbzE, RT-PCR analysis of total RNA isolated from a 3-hydroxybenzoate-grown culture of strain NCIMB 9867 was carried out using a primer pair, FE-RT-F (5=-GGC AGA ACG CCA ATA CC A-3=) and FE-RT-R (5=-GCA ACA CCG AAC GCA ACG-3=), that spanned across the hbzF and hbzE genes.…”

mentioning

confidence: 99%

HbzF Catalyzes Direct Hydrolysis of Maleylpyruvate in the Gentisate Pathway of Pseudomonas alcaligenes NCIMB 9867

Liu

Zhou

2013

Appl Environ Microbiol

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bHbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

show abstract

Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867

Cited by 14 publications

References 26 publications

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

HbzF Catalyzes Direct Hydrolysis of Maleylpyruvate in the Gentisate Pathway of Pseudomonas alcaligenes NCIMB 9867

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