Difference between revisions of "Part:BBa K2346000"
 
(3 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2346000 short</partinfo>
 
<partinfo>BBa_K2346000 short</partinfo>
  
−
From Agrobacterium tumefaciens. The enzyme contains two copper center, namely CuA and Cuz, which can transfer the electron.
+
Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). We are still working on that. Since nitric oxide is nearly insoluable in water, it’s hard for us to create a aquatic reaction system for assaying the enzyme reactivity. We are still working on that. But currently we plan to create a reaction environment in a sealed container filled with solution, enzyme, and nitric oxide gas, By constant shake over, let’s say, two hours, we’ll then measure the change in the concentration of NO using the Gas-Phase Molecular Absorption Spectrometry to see if the presence of the enzyme made any difference on the concentration.
−
 
+
−
The assay:
+
−
We prepared a standard measuring mixture(2ml) consisting of 50mM Tris-HCL,pH9, 0.5mM methyl viologen, a proper amount of NosZ and 1mM sodium dithionite. Methyl viologen and sodium dithionite will go on to react and form a blue intermediate product, with a relativelly stable absorbance at 600nm. The blue interdiate product actually serves as an electron donor for the reduction of nitrous oxide. Therefore, as the reaction goes on, the blue substance starts to lose electrons and its color dims, resulting in a lower 600nm absorbance. Thus, by directly measuring the change of color in the system, we are able to track the processing of the reaction.  
+
−
 
+
  
  
Line 22: Line 18:
 
<partinfo>BBa_K2346000 parameters</partinfo>
 
<partinfo>BBa_K2346000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
<h2>Background</h2>
 +
 +
Cytochrome P450 55B1 from Chlamydomonas reinhardtii<br>
 +
General Equation: $ \ce{2NO + NAD(P)H + H+ → N2O + NADP+ + H2O} $
 +
 +
[[Image:/wiki/images/6/6c/T--HFLS_H2Z_Hangzhou--img_enzyme_nor1.png]]
 +
 +
We found three assay methods to test the activity of nitric oxide reductase: using NO measurement Kit, titration and Gas-Phase Molecular Absorption Spectrometry. The first two methods were failed, and the details and reasons were recorded below. Due to limitations of lab equipment, we were not able to conduct the experiment using the third method.
 +
 +
 +
[[Image:/wiki/images/6/64/T--HFLS_H2Z_Hangzhou--img_enzyme_nor2.png]]
 +
<div class="img-name">Nitric oxide reductase's tertiary structure</div>
 +
 +
<h2>Assay method 1: NO Measurement Kit</h2>
 +
 +
The NO kit we planned to use was developed by Nanjing Jiancheng Bioengineering Institute, and the concentration of NO in the solution can be calculated by the concentration of $\ce{NO3-}$, because NO will be reduced to $\ce{NO3-}$ in blood plasma of chicken or mice. We prepared the solution according to the method as below:
 +
<br><br>
 +
<table class="frameless ref-table">
 +
<tbody>
 +
<tr>
 +
<td><div class="no">1</div></td>
 +
<td>Remove the air in the flask by passing through nitrogen gas for 30 minutes.</td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">2</div></td>
 +
<td>Pass through nitric oxide gas for 45 minutes in Flask C that contains ddH2O.</td>
 +
</tr>
 +
 +
<tr>
 +
<td><div class="no">3</div></td>
 +
<td>Flask A contains 30% H2O2 solution, and Flask B contains KMnO4 / NaOH solution, to oxidize and absorb the undissolved gas.[1]</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br>
 +
However, although the NO solution we prepared using this method was saturated, the concentration was too low for the verification to proceed.
 +
 +
[[Image:/wiki/images/5/59/T--HFLS_H2Z_Hangzhou--img_enzyme_nor3.png]]
 +
<div class="img-name">Fig 1. Setup of equipments for preparing NO solution</div>
 +
 +
[[Image:/wiki/images/5/5a/T--HFLS_H2Z_Hangzhou--img_enzyme_nor4.jpg]]
 +
<div class="img-name">Fig 2. NO gas container</div>
 +
 +
[[Image:/wiki/images/1/19/T--HFLS_H2Z_Hangzhou--img_enzyme_nor5.png]]
 +
<div class="img-name">Fig 3. Equipments setup</div>
 +
 +
<h2>Assay Method 2: Titration</h2>
 +
 +
By measuring the concentration of nitrous oxide—the final product of the reduction of nitric oxide, the amount of nitric oxide reacted can be calculated, and therefore the activity of nitric reductase can be verified. When the reaction is complete, acidic potassium permanganate is added to the solution, and the highly oxidative potassium permanganate reacts with nitrous oxide. We then use oxalic acid to titrate the remaining potassium permanganate. Therefore, the amount of potassium permanganate used in the reaction with nitrous oxide can be calculated, and the amount of nitrous oxide produce can be found.
 +
 +
<br><br>
 +
 +
However, even for the most stable solution of nitrous oxide—the standard solution of 1.00mg/ml, nitrous oxide will escape in gas form, and the escaping speed is not constant. As a result, the method of directly measuring the amount of nitrous oxide is not valid.
 +
 +
<img class="framed img" src="/img/enzyme/nor6.png"></img>
 +
<div class="img-name">Fig 4. Standard N2O solution</div>
 +
 +
<h2>Assay Method 3: Gas-Phase Molecular Absorption Spectrometry</h2>
 +
 +
The turnover of the overall reaction of nitric oxide reductase was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N2 mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). Unfortunately, since we could not find access to the required gas divider, we were not able to verify the results. The results from the original paper was shown in Fig 4.
 +
<br>
 +
 +
<h3>Protocol</h3>
 +
<table class="frameless ref-table">
 +
<tbody>
 +
<tr>
 +
<td><div class="no">1</div></td>
 +
<td>Prepare the buffer: 0.1 M sodium phosphate at pH 7.2.</td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">2</div></td>
 +
<td>Add reduced form of NADH.</td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">3</div></td>
 +
<td>Purge the air in the buffer solution by nitrogen gas to prevent formation of NO2 before adding NO.</td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">4</div></td>
 +
<td>Introduce NO gas into the sample solution after passing 1 M KOH and the buffer solutions. </td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">5</div></td>
 +
<td>Incubate the buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM) in the optical cell at 10°C for 5 min.</td>
 +
</tr>
 +
<tr>
 +
<td><div class="no">6</div></td>
 +
<td>Measure the absorbance (340nm) of the solutions.</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 +
<h2>Results</h2>
 +
 +
Due to limited access to equipments, we could not directly measure the reactivity of NOR. However, experiments had been carried out for the same enzyme expressed also expressed in E.coli, with method listed above. For the continuation of our project, we had to draw conclusion from other’s experiment results.
 +
 +
[[Image:/wiki/images/7/74/T--HFLS_H2Z_Hangzhou--img_enzyme_nor7.png]]
 +
<div class="img-name">Fig 5. Time course of the absorption change of the reduced form of NADH at 340 nm in the NO reduction reaction catalyzed by P450nor. [2]</div>
 +
 +
<h2>Conclusion</h2>
 +
 +
<b>We delightedly found out that the curve became smooth and parallel to the x-axis just within the course of five minutes. This represented nearly all the substrates had been converted to products in an extremely rapid rate: much more rapid than nitrite reductase. This means that whenever NO is released from nitrite reductase as product, NOR can utilize it as substrate quickly.</b>
 +
 +
<h2>Reference</h2>
 +
<table class="frameless ref-table">
 +
<tbody>
 +
<tr>
 +
<td><div class="no noind">1</div></td>
 +
<td>程得胜, 黄曜, 黄郁芳. 一氧化氮标准储备水溶液浓度的测定及稳定性分析[J]. 分析测试学报, 2007, 26(4):566-569.</td>
 +
</tr>
 +
 +
<tr>
 +
<td><div class="no noind">2</div></td>
 +
<td>Shiro Y., Fujii M., Iizuka T., Adachi S., Tsukamoto K., Nakahara K., Shoun H. 1995. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617–162310.1074/jbc.270.4.1617.</td>
 +
</tr>
 +
 +
<tr>
 +
<td><div class="no noind">3</div></td>
 +
<td>Shoun H, Fushinobu S, Jiang L, et al. Fungal denitrification and nitric oxide reductase cytochrome P450nor[J]. Philosophical Transactions of the Royal Society of London, 2012, 367(1593):1186.</td>
 +
</tr>
 +
</tbody>
 +
</table>

Latest revision as of 01:47, 2 November 2017


Nitric oxide reductase (NOR)

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). We are still working on that. Since nitric oxide is nearly insoluable in water, it’s hard for us to create a aquatic reaction system for assaying the enzyme reactivity. We are still working on that. But currently we plan to create a reaction environment in a sealed container filled with solution, enzyme, and nitric oxide gas, By constant shake over, let’s say, two hours, we’ll then measure the change in the concentration of NO using the Gas-Phase Molecular Absorption Spectrometry to see if the presence of the enzyme made any difference on the concentration.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
    Illegal XhoI site found at 1216
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1200
  • 1000
    COMPATIBLE WITH RFC[1000]


Background

Cytochrome P450 55B1 from Chlamydomonas reinhardtii
General Equation: $ \ce{2NO + NAD(P)H + H+ → N2O + NADP+ + H2O} $

File:/wiki/images/6/6c/T--HFLS H2Z Hangzhou--img enzyme nor1.png

We found three assay methods to test the activity of nitric oxide reductase: using NO measurement Kit, titration and Gas-Phase Molecular Absorption Spectrometry. The first two methods were failed, and the details and reasons were recorded below. Due to limitations of lab equipment, we were not able to conduct the experiment using the third method.


File:/wiki/images/6/64/T--HFLS H2Z Hangzhou--img enzyme nor2.png

Nitric oxide reductase's tertiary structure

Assay method 1: NO Measurement Kit

The NO kit we planned to use was developed by Nanjing Jiancheng Bioengineering Institute, and the concentration of NO in the solution can be calculated by the concentration of $\ce{NO3-}$, because NO will be reduced to $\ce{NO3-}$ in blood plasma of chicken or mice. We prepared the solution according to the method as below:

<tbody> </tbody>
1
 Remove the air in the flask by passing through nitrogen gas for 30 minutes.
2
 Pass through nitric oxide gas for 45 minutes in Flask C that contains ddH2O.
3
 Flask A contains 30% H2O2 solution, and Flask B contains KMnO4 / NaOH solution, to oxidize and absorb the undissolved gas.[1]


However, although the NO solution we prepared using this method was saturated, the concentration was too low for the verification to proceed.

File:/wiki/images/5/59/T--HFLS H2Z Hangzhou--img enzyme nor3.png

Fig 1. Setup of equipments for preparing NO solution

File:/wiki/images/5/5a/T--HFLS H2Z Hangzhou--img enzyme nor4.jpg

Fig 2. NO gas container

File:/wiki/images/1/19/T--HFLS H2Z Hangzhou--img enzyme nor5.png

Fig 3. Equipments setup

Assay Method 2: Titration

By measuring the concentration of nitrous oxide—the final product of the reduction of nitric oxide, the amount of nitric oxide reacted can be calculated, and therefore the activity of nitric reductase can be verified. When the reaction is complete, acidic potassium permanganate is added to the solution, and the highly oxidative potassium permanganate reacts with nitrous oxide. We then use oxalic acid to titrate the remaining potassium permanganate. Therefore, the amount of potassium permanganate used in the reaction with nitrous oxide can be calculated, and the amount of nitrous oxide produce can be found.



However, even for the most stable solution of nitrous oxide—the standard solution of 1.00mg/ml, nitrous oxide will escape in gas form, and the escaping speed is not constant. As a result, the method of directly measuring the amount of nitrous oxide is not valid.

<img class="framed img" src="/img/enzyme/nor6.png"></img>

Fig 4. Standard N2O solution

Assay Method 3: Gas-Phase Molecular Absorption Spectrometry

The turnover of the overall reaction of nitric oxide reductase was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N2 mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). Unfortunately, since we could not find access to the required gas divider, we were not able to verify the results. The results from the original paper was shown in Fig 4.

Protocol

<tbody> </tbody>
1
 Prepare the buffer: 0.1 M sodium phosphate at pH 7.2.
2
 Add reduced form of NADH.
3
 Purge the air in the buffer solution by nitrogen gas to prevent formation of NO2 before adding NO.
4
 Introduce NO gas into the sample solution after passing 1 M KOH and the buffer solutions.
5
 Incubate the buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM) in the optical cell at 10°C for 5 min.
6
 Measure the absorbance (340nm) of the solutions.

Results

Due to limited access to equipments, we could not directly measure the reactivity of NOR. However, experiments had been carried out for the same enzyme expressed also expressed in E.coli, with method listed above. For the continuation of our project, we had to draw conclusion from other’s experiment results.

File:/wiki/images/7/74/T--HFLS H2Z Hangzhou--img enzyme nor7.png

Fig 5. Time course of the absorption change of the reduced form of NADH at 340 nm in the NO reduction reaction catalyzed by P450nor. [2]

Conclusion

We delightedly found out that the curve became smooth and parallel to the x-axis just within the course of five minutes. This represented nearly all the substrates had been converted to products in an extremely rapid rate: much more rapid than nitrite reductase. This means that whenever NO is released from nitrite reductase as product, NOR can utilize it as substrate quickly.

Reference

<tbody> </tbody>
1
 程得胜, 黄曜, 黄郁芳. 一氧化氮标准储备水溶液浓度的测定及稳定性分析[J]. 分析测试学报, 2007, 26(4):566-569.
2
 Shiro Y., Fujii M., Iizuka T., Adachi S., Tsukamoto K., Nakahara K., Shoun H. 1995. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617–162310.1074/jbc.270.4.1617.
3
 Shoun H, Fushinobu S, Jiang L, et al. Fungal denitrification and nitric oxide reductase cytochrome P450nor[J]. Philosophical Transactions of the Royal Society of London, 2012, 367(1593):1186.