Transformation and Mineralization of Aroclor 1254

Köhler, Hans-Peter; Kohler-Staub, D.; Focht, D. D.

doi:10.1007/978-1-4899-0824-7_43

Cited by 9 publications

(13 citation statements)

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“…P. azelaica ' HBP1 is able to use 2-HBP and 2,2h-diHBP as a sole source of carbon and energy (Kohler et al, 1988). ' P. azelaica ' HBP104 (Jaspers et al, 2000) and HBP117 originate from strain HBP1 and contain luxAB-based transcriptional fusions with the upstream regions of hbpC and hbpR, respectively, integrated on the chromosome in monocopy.…”

Section: Methodsmentioning

confidence: 99%

“…The hbp system allows ' Pseudomonas azelaica ' HBP1 to metabolize 2-hydroxybiphenyl (2-HBP) and 2,2h-diHBP by employing a meta-cleavage pathway (Kohler et al, 1988(Kohler et al, , 1993Schmid, 1997). The hbp system consists of the two transcriptional units, hbpCA and hbpD, encoding the enzymes for the first steps of 2-HBP degradation, plus the regulatory gene hbpR, which encodes the transcriptional activator (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’ HBP1

2001

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' Pseudomonas azelaica ' HBP1 degrades 2-hydroxybiphenyl (2-HBP) and 2,2'-diHBP by employing a meta-cleavage pathway encoded by the hbpCAD genes. The regulatory gene hbpR, located directly upstream of the hbpCAD genes and oriented in the opposite direction, encodes a transcription activator protein belonging to the so-called XylR/DmpR subclass within the NtrC family. HbpR activates transcription from two separate σ 54 -dependent promoters upstream of the hbpC and the hbpD genes, in the presence of the pathway substrates 2-HBP and 2,2'-diHBP. The DNA region upstream of the hbpC gene displays an unusual organization, containing two adjacent 03 kb regions that share 71 % sequence identity. The DNA region most proximal to the hbpC promoter harbours one pair of putative upstream activating sequences (UASs C-1/C-2) and a small cryptic ORF that shows homology to hbpR itself. The second, more distal, region contains a second pair of putative UASs (UASs C-3/4) and the 5'-part of the hbpR gene. Transcriptional fusions in Escherichia coli between different deletions of the hbpR-hbpC intergenic region and the genes for bacterial luciferase revealed that most if not all of the transcriptional output from the hbpC promoter is mediated from the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 were deleted and UASs C-3/C-4 were placed in an appropriate position with respect to the promoter region, the hbpC promoter was still inducible with 2-HBP, albeit at a lower level. Transcription studies in E. coli and ' P. azelaica ' revealed that the divergently oriented hbpR gene is expressed constitutively from a σ 70 -dependent promoter situated within the cryptic ORF. The presence of UAS pair C-3/C-4 mediated a slightly higher promoter activity for transcription of hbpR.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’ HBP1

2001

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“…2-Hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 is a flavoprotein aromatic hydroxylase that catalyzes the hydroxylation of a variety of 2-substituted phenols to the corresponding catechols (12)(13)(14). The mechanism of HbpA has been extensively studied by spectroscopic techniques, which revealed that molecular oxygen is activated via the formation of a flavin (C4a)-hydroperoxide (15), a common intermediate in the reaction cycle of this enzyme family (16).…”

mentioning

confidence: 99%

“…The mechanism of HbpA has been extensively studied by spectroscopic techniques, which revealed that molecular oxygen is activated via the formation of a flavin (C4a)-hydroperoxide (15), a common intermediate in the reaction cycle of this enzyme family (16). HbpA has a broad substrate spectrum, but does not hydroxylate indole (13,17). By directed enzyme evolution, we recently changed the substrate reactivity of HbpA toward 2-tert-butylphenol, a substrate that is not converted by the wild-type enzyme (18,19).…”

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confidence: 99%

Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

Meyer

Würsten

Schmid

et al. 2002

Journal of Biological Chemistry

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“…HbpA was first found in Pseudomonas azelaica HBP1, a soil bacterium that is able to grow on the fungicide 2-hydroxybiphenyl as sole source of carbon and energy (7). HbpA catalyzes the ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl, which is then converted to 2-hydroxy-6-phenyl-6-oxo-2,4-hexadienoic acid by a meta ring cleavage dioxygenase (HbpC).…”

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confidence: 99%

Changing the Substrate Reactivity of 2-Hydroxybiphenyl 3-Monooxygenase from Pseudomonas azelaica HBP1 by Directed Evolution

Meyer¹,

Schmid²,

Held³

et al. 2002

Journal of Biological Chemistry

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The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased V max from 0.22 to 0.43 units mg ؊1 protein and decreased the K m from 588 to 143 M, improving k cat /K m by a factor of 8.2. With 2-tert-butylphenol as the substrate, k cat was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K m for guaiacol decreased with this mutant, but the K m for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1% sequence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile 244 of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val 368 and Leu 417 are not close to the active site and would not have been obvious candidates for modification by rational design.

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Transformation and Mineralization of Aroclor 1254

Cited by 9 publications

References 0 publications

Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’ HBP1

Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’ HBP1

Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

Changing the Substrate Reactivity of 2-Hydroxybiphenyl 3-Monooxygenase from Pseudomonas azelaica HBP1 by Directed Evolution

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