Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase

Oppenheimer, Michelle; Pierce, Brad S.; Crawford, Joshua A.; Ray, Keith; Helm, Richard F.; Sobrado, Pablo

doi:10.1016/j.abb.2010.02.006

Cited by 11 publications

(12 citation statements)

References 35 publications

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“…To further enhance the oxidation activity of the engineered E. coli cells, it would be important to achieve high-level and soluble expression of MimB. It has recently been reported that fusion of the reductase component of tetrahydrofuran monooxygenase to maltose binding protein markedly increased the solubility of this component (24). Similar to this, fusion of MimB to an appropriate tag sequence might lead to its high-level soluble expression.…”

Section: Discussionmentioning

confidence: 86%

“…4). It was reported that the reductase components of binuclear iron monooxygenases that contain [Fe-S] centers were amenable to inactivation in E. coli cells, particularly under high-level-expression conditions (15,24,32). For example, the reductase component of toluene 4-monooxygenase has been functionally expressed under the control of a weak promoter (32).…”

Section: Discussionmentioning

confidence: 99%

“…The transformed E. coli Rosetta 2(DE3)pLysS cells were cultivated at 30°C in LB medium supplemented with chloramphenicol (34 g/ml), kanamycin (15 g/ml), streptomycin (50 g/ml), and/or ampicillin (50 g/ml). When the cell growth reached an optical density at 600 nm (OD 600 ) of 0.6 to 0.8, ferric citrate (100 g/ml), ammonium ferric citrate (100 g/ml), and L-cysteine (0.2 mM) were added to the medium to supply iron and sulfur for an iron protein (i.e., MimA) and an iron-sulfur protein (i.e., MimB) (23,24). Isopropyl-␤-D-thiogalactopyranoside (IPTG, 0.1 mM) was also added to the medium, and cultivation was continued at 25°C for an additional 15 h. Cells were harvested by centrifugation at 15,000 ϫ g for 10 min at 4°C, washed with potassium phosphate buffer (50 mM, pH 7.5) containing glycerol (10%, vol/vol), and stored at Ϫ80°C until use.…”

Section: Methodsmentioning

confidence: 99%

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Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Furuya

Hayashi

Kino

2013

Appl Environ Microbiol

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Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Furuya

Hayashi

Kino

2013

Appl Environ Microbiol

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“…It is well known that cofactors are often lost during heterologous expression. Indeed, the expression of iron-sulfur cluster-containing proteins has been reported to be difficult, and a significant amount of expressed proteins have been found to lack their iron-sulfur cluster (Oppenheimer et al, 2010). Assuming that some portion of recombinant HCAR was denatured and had lost the iron, holo-HCAR might potentially have more iron per protein moiety.…”

Section: The Redox Cofactors Of Hcarmentioning

confidence: 99%

Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis

et al. 2011

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The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.

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“…A gene cluster responsible for THF degradation in Pseudonocardia strain K1 was reported by Thiemer et al (2003) and Oppenheimer et al (2010). The genus Rhodococcus was the first one published to have the capability of utilizing THF as sole carbon source.…”

Section: Introductionmentioning

confidence: 99%

Evidence for horizontal gene transfer of a tetrahydrofuran monooxygenase: Cloning and analysis of a gene cluster for tetrahydrofuran degradation in Rhodococcus sp. YYL

Yao¹,

Lü²,

Zhu³

et al. 2013

Afr. J. Microbiol. Res.

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A gene cluster encoding tetrahydrofuran (THF) degrading related enzymes was first isolated from Rhodococcus sp. YYL, a strain of the genus Rhodococcus, which was first reported to degrade tetrahydrofuran, by combination of COnsensus-DEgenerate Hybrid oligonucleotide Primer PCR and thermal asymmetric interlaced PCR. Sequence analysis of the gene cluster revealed 9 open reading frames containing genes encoding a Rhodococcus sp. YYL multiple component tetrahydrofuran monooxygenase. The tetrahydrofuran monooxygenase genes had similarities higher than 92% with these in Pseudonocardia sp. K1, but had low similarities with other monooxygenase genes using BLAST search in GenBanK. The high specific of tetrahydrofuran monooxygenase genes was further proved by PCR amplification of no product using genomic DNA of other bacteria or even activated sludge as template with primers targeting at the α-subunit of this monooxygenase. Phylogenetic analysis based on amino acid sequences and 16S rDNA strongly suggested that this monooxygenase occurred by horizontal gene transfer. Base composition analysis and the existence of a transposase gene in the gene cluster further proposed that this monooxygenase originated from the genus Rhodococcus and it might be transformed by a transposon between different species.

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Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase

Cited by 11 publications

References 35 publications

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis

Evidence for horizontal gene transfer of a tetrahydrofuran monooxygenase: Cloning and analysis of a gene cluster for tetrahydrofuran degradation in Rhodococcus sp. YYL

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