 |
| B. stearothermophilus, cmapspublic3.ihmc.us |
Geobacillus stearothermophilus is a thermophile, rodshaped, gram-positive bacteria. It's growing temperature is about 30-75 degrees C, can be widely found in various warm environment such as, sand, ocean, hot springs. It is capable of anaerobic respiration.
NOR is a membrane-integrated protein that contains iron-containing enzymes involved in anaerobic respiration. The enzyme is necessary for one step of the sequencial reduction of NO3- > NO2- > NO > N2O > N2. It catalyses the reduction of NO to N2O by breaking the N-O bond and forming N-N bond using two protons and two electrons.
(2NO + 2H+ + 2e- -> N2O + H2O)
NOR is a membrane-integrated protein that contains iron-containing enzymes involved in anaerobic respiration. The enzyme is necessary for one step of the sequencial reduction of NO3- > NO2- > NO > N2O > N2. It catalyses the reduction of NO to N2O by breaking the N-O bond and forming N-N bond using two protons and two electrons.
(2NO + 2H+ + 2e- -> N2O + H2O)
NOR is studied for:
- Environmental sciences: N2O is a major greenhouse gas, 310x stronger than carbon. Understanding its formation in nature may help to counter its accumulation.
- Medicine: some NORs are expressed in the absence of other denitrifying enzymes in pathogenic bacteria, hinting that its role is not for energy production through coupling, but instead for neutralising the highly toxic NO radical produced by the macrophages. Better treatment may be devised if this defense can be countered.
The NOR and COX (cytochrome
oxidase) evolved from the same ancestor protein, they have a structural similarity
and metal ligands. They both classified as the heme-copper oxidase (HCO)
family. There are two subgroups of NOR, namely qNOR and cNOR. How the proteins are related yet was still unknown due to the previously unavailabile of qNOR structural data.
The reference paper comes in to the picture, as its successful crystalisation of qNOR helps to provide the much needed missing link.
Steps leading to crystallisation of qNOR are listed below:
The reference paper comes in to the picture, as its successful crystalisation of qNOR helps to provide the much needed missing link.
Steps leading to crystallisation of qNOR are listed below:
- Construction of qNOR recombinant expression plasmid
- pCR2.1-TOPO vector by Invitrogen was used to clone qNOR gene-containing fragment for sequencing and identification of norZ ORF.
- ORF-containing fragment was amplified and inserted in pET-22b vector, between NdeI and XhoI sites, downstream of a T7 promoter
- Plasmid was transformed into Rosseta2, an E. coli strain. This strain has T7 polymerase gene under Lac promoter
- Purification of recombinant qNOR
- IPTG was used to induce expression.
- Solubilisation of qNOR was done using βOG detergent.
- Affinity chromatography was done using Ni-NTA agarose coloumn
- Crystallisation and diffraction analysis
- Done using sitting drop vapor diffusion.
- Phases were calculated by MAD (Multiwavelength Anomalous Diffraction) method using SHARP and SOLOMON
- Replacement of Sulphur to Selenium in methionine residues to prompt diffraction data differences, which helps to calculate the initial phases.
- His-tag was not removed during crystallisation, as it doesn't interfere native protein folding.
 |
UniProt of qNOR from Grobacillus stearothrtmophillus
|
