Dataset Information

Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Comamonas sp. Expressed in Escherichia coli.

ABSTRACT: 2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBDO) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the bphC gene from Comamonas sp. SMN4, which encodes 2,3-DBDO with His-tag, was cloned into a plasmid pQE30 in E. coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified active 2,3-DBDO showed a single band around 33 kDa, corresponding the molecular mass of 2,3-DBDO subunit. Two fractions around 170 and 100 kDa were separated in gel filtration chromatography, but only former one (the fraction of 170 kDa) has 2,3-DBDO activity. The 2,3-DBDO was reported as the polymeric protein consisted of eight subunits. However, the fraction corresponding octameric protein of 2,3-DBDO was not found in the gel filtration chromatography. The 2,3-DBDO was exhibited the maximum activity at pH 9.0 and was stable at pH 8.0, relatively. The circular dichroism (CD) data showed that 2,3-DBDO had an ?-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. However, this high stable folding structure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The enzyme was thermally stable and active up to 40 °C. The conformational data by CD spectra were consistent with the stability of 2,3-DBDO by checking the activity. The binding affinity (Km ) for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7, 24 ?M, 50 mM and 625 ?M, respectively.

SUBMITTER: Lee N

PROVIDER: S-EPMC5061692 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Comamonas sp. Expressed in Escherichia coli.

Lee Nari N Kwon Dae Yong DY

Indian journal of microbiology 20160521 4

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBDO) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the bphC gene from Comamonas sp. SMN4, which encodes 2,3-DBDO with His-tag, was cloned into a plasmid pQE30 in E. coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified active 2,3-DBDO showed a single band around 33 kDa, corresponding the molecular mass of 2,3-DBDO subunit. ...[more]

PMID: 27784944

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Project description:The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. FeII is required for the catalytic activity of the purified enzyme. The apparent Km and Vmax values of MabB for 5ASA are 52.0 ± 5.6 ?M and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (?70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.

Project description:Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus COMAMONAS: Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3' end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Km(r) cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of sigma(54)-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-beta-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

Dataset Information

Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Comamonas sp. Expressed in Escherichia coli.

Publications

Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from <i>Comamonas</i> sp. Expressed in <i>Escherichia coli</i>.

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