A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Chen, Ying; Hunsicker-Wang, L.M.; Pacoma, Ronald L.; Luna, Eugene; Fee, James A.

doi:10.1016/j.pep.2004.11.014

Cited by 33 publications

(53 citation statements)

References 55 publications

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“…The virus vector allows for the stable production of proteins in large-scale fermentations of S. solfataricus, which might be of interest for industrial applications when the system is further improved. While a few studies have recently demonstrated the expression of proteins in hyperthermophilic bacteria, in particular in Thermus thermophilus (8,21), this system will allow the production of proteins from hyperthermophilic archaea.…”

Section: Discussionmentioning

confidence: 99%

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Albers

Jonuscheit

Dinkelaker

et al. 2006

Appl Environ Microbiol

119

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The homologous and heterologous expression of genes is a prerequisite for most biochemical studies of protein function. A vast variety of systems have been developed for protein production in members of the Bacteria and Eukarya, using numerous combinations of vector and promoter systems. Members of the Archaea, the third domain of life, are much less amenable to genetic manipulation. Transformation tools for the production of recombinant proteins exist for only a few species (for a review, see reference 2). A shuttle vector has been described for the expression of bacterial and methanococcal genes in Methanococcus maripaludis (12). Heterologous expression of bacterial and eukaryotic genes as well as of homologous genes has been achieved in the genetically most accessible archaea, the mesophilic, salt-dependent Halobacterium spp. (14,17,33). However, no such system has existed so far for thermophilic or hyperthermophilic archaea. Mesophilic hosts, in particular Escherichia coli, have been used to produce thermostable proteins for biochemical characterization and crystallographic studies (e.g., see references 22, 23, 29, and 34). However, a considerable number of proteins of hyperthermophiles fold into their native state only under natural conditions of high temperature or in the presence of their native cofactors. Furthermore, the production of recombinant and tagged proteins in native thermophilic hosts allows the identification of associated factors or even larger protein complexes.The crenarchaeote Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. It grows optimally at 80°C and pH 3 under aerobic and heterotrophic conditions. Since many studies on the transcription, translation, and replication of this extremophile have been performed in vitro (4,5,10,40), it is highly desirable to develop genetic tools for in vivo studies and for high-level production of proteins in this organism. Initial transformation systems and selectable markers have been established for Sulfolobus solfataricus in some laboratories (3,6,7,9,16,41). We have recently developed a reporter gene system based on the virus SSV1 as well as the selectable marker genes pyrEF for the complementation of uracil auxotrophic mutants. The latter genes allow stabilization of the propagation of the vector by growing transformants under selective conditions (16). Moreover, strong, heat-inducible expression of the reporter gene lacS, coding for ␤-galactosidase, was demonstrated when it was placed under the control of the promoter of the major heat shock chaperonin gene tf55␣ (16).In this study, we have used and improved this vector system for the heterologous and homologous expression of genes in S. solfataricus. We have constructed a set of entry vectors and introduced a transcriptional terminator and a second inducible promoter. Our system allows for the high-level production of functional and tagged cytoplasmic and membrane-associated

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Section: Discussionmentioning

confidence: 99%

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Albers

Jonuscheit

Dinkelaker

et al. 2006

Appl Environ Microbiol

119

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“…Protein expression, purification, and steady-state activity measurements were as previously described (48). Proton pumping was measured following reconstitution of the purified oxidase into proteoliposomes (7) and measurement of the acidification of the external solution concomitant with a known number of turnovers using cytochrome c 552 as the electron donor.…”

Section: Methodsmentioning

confidence: 99%

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba₃fromThermus thermophilus

Chang¹,

Choi

Vakkasoglu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba 3 -type oxygen reductase from Thermus thermophilus , leading from the propionates of heme a 3 to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a 3 , and for Glu126 II (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126 II , and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a 3 , was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a 3 or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a 3 is a good candidate to be the proton loading site.

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“…The expression, purification, and characterization of the T. thermophilus ba 3 oxygen reductase was performed as previously described (39). T. thermophilus cytochrome c 552 also was prepared as previously described (40).…”

Section: Methodsmentioning

confidence: 99%

The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

Chang¹,

Hemp

Chen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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102

185

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The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B-and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D-and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba 3 oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O 2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidinetyrosine, the Cu B and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.cytochrome c oxidase ͉ respiratory enzymes

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A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cited by 33 publications

References 55 publications

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba₃fromThermus thermophilus

The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

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A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cited by 33 publications

References 55 publications

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3fromThermus thermophilus

The cytochrome ba 3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

Contact Info

Product

Resources

About

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba₃fromThermus thermophilus

The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping