Difference between revisions of "Part:BBa K808014"
 
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AroY is reported to catalyze the reaction of protocatechuate (3,4-Dihydroxybenzoate) to catechol under anaerobic conditions. This is the last step in the degradation of terephtalic acid to catechol. Gel permeation chromatography shows that AroY has a homopentomeric structure and a mass of 285.4 kDa (Figure 2).
 
AroY is reported to catalyze the reaction of protocatechuate (3,4-Dihydroxybenzoate) to catechol under anaerobic conditions. This is the last step in the degradation of terephtalic acid to catechol. Gel permeation chromatography shows that AroY has a homopentomeric structure and a mass of 285.4 kDa (Figure 2).
  
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[[Image:GPC_AroY.JPG|300px|thumb|right|Figure 2. '''GPC analysis of AroY'''. The Peak of AroY has a retention time of 33 minutes.]]
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[[Image:GPC_AroY.JPG|500px|thumb|left|Figure 2. '''GPC analysis of AroY'''. The Peak of AroY has a retention time of 28 minutes.]]
  
  
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==Part Characterisation ==
 
==Part Characterisation ==
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[[Image:AroY_ff.jpg|thumb|right|300px|Figure 3. '''Functional test for AroY:'''<br> '''1)''' AroY with 50 mM protocatechuate after 24 h; <br>'''2)''' After addition of [https://parts.igem.org/Part:BBa_K316003 XylE] and 10 min incubation]]
  
  
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We incubated AroY with 50 mM protocatechuic acid under anaerobic conditions. The anaerobic conditions are important for enzymatic activity, as AroY is sensitive to exposure to oxygen. During incubation a development of gas could be seen and after an incubation for 24 hours a brown colour typical for 1,2-Benzoquinone could be observed. The following incubation with [https://parts.igem.org/Part:BBa_K316003 XylE] produced quickly the typical colour for thereaction of catechol to 2-hydroxymuconate semialdehyde (Figure 3). For further information, please visit the [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#Enzyme_assays iGEM TU Darmstadt 2012 wiki page].
  
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[[Image:AroY_ff.jpg|thumb|right|300px|Figure 3. '''Functional test for AroY:'''<br> '''1)''' AroY with 50 mM Protocatechuate after 24 h; <br>'''2)''' after addition of XylE and 10 min incubation]]
 
  
  

Latest revision as of 02:50, 27 September 2012

AroY: Catalyzes protocatechuate to catechol

Figure 1. Gaussian network modelling of AroY. The supposed membrane anchor in red. For simulation parameters http://2012.igem.org/Team:TU_Darmstadt/Modeling_GNM#AroY click here.

AroY codes for the protocatechuate deacarboxylase from Klebsiella pneumonie ssp. pneumonie. It has a molecular mass of 57 kDa and is reported to form a complex of five identical AroY subunits. [http://2012.igem.org/Team:TU_Darmstadt/Modeling_GNM#AroY Gaussian network modelling] showed a flexible helix linker. This could be either a membrane anchor or responsible for the formation of a pentamer.


Usage and Biology

AroY is reported to catalyze the reaction of protocatechuate (3,4-Dihydroxybenzoate) to catechol under anaerobic conditions. This is the last step in the degradation of terephtalic acid to catechol. Gel permeation chromatography shows that AroY has a homopentomeric structure and a mass of 285.4 kDa (Figure 2).

Figure 2. GPC analysis of AroY. The Peak of AroY has a retention time of 28 minutes.










Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 309
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 37



Part Characterisation

Figure 3. Functional test for AroY:
1) AroY with 50 mM protocatechuate after 24 h;
2) After addition of XylE and 10 min incubation


We incubated AroY with 50 mM protocatechuic acid under anaerobic conditions. The anaerobic conditions are important for enzymatic activity, as AroY is sensitive to exposure to oxygen. During incubation a development of gas could be seen and after an incubation for 24 hours a brown colour typical for 1,2-Benzoquinone could be observed. The following incubation with XylE produced quickly the typical colour for thereaction of catechol to 2-hydroxymuconate semialdehyde (Figure 3). For further information, please visit the [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#Enzyme_assays iGEM TU Darmstadt 2012 wiki page].






References

  • He, Z. and J. Wiegel, Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum. J Bacteriol, 1996. 178(12): p. 3539-43.
  • Grant, D.J. and J.C. Patel, The non-oxidative decarboxylation of p-hydroxybenzoic acid, gentisic acid, protocatechuic acid and gallic acid by Klebsiella aerogenes (Aerobacter aerogenes). Antonie Van Leeuwenhoek, 1969. 35(3): p. 325-43.