Part:BBa_K2423008

UGTCs2 with BBa_J04500

UGTCs2 is an enzyme that catalyzes the conversion of crocetin to crocin. This is the third and final step in the zeaxanthin to crocin pathway. In nature this enzyme can be found in Crocus Sativus. Crocus Sativus is the plant where saffron is harvested from. This BioBrick is verified by sequencing and a homology model was created, which was used in a 100 ns molecular dynamics simulation.

The gene of interest in this part is regulated by BBa_J04500. This implies that it has to be induced by lactose or any of its closely related derivates, such as IPTG.

This BioBrick is an improved version of BBa_K1033112 and a slightly modified version of BBa_K2423002 (different promoter and RBS).

For more information read our wiki found here: http://2017.igem.org/Team:Uppsala.

Usage and Biology

Saffron, a well recognized but expensive spice, has not only uses in terms of cooking but compounds found in saffron have been shown to help with inflammation (1), neurodegenerative diseases (2) and more. Some of those compounds namely zeaxanthin, crocetin dialdehyde, crocetin and crocin are all a part of the same metabolic pathway in the plant specie Crocus Sativus. Not only are these compounds in saffron helpful in terms of their potential medicinal properties, but also the fact that they are very colorful makes them interesting as organic dyes for industrial purposes. These aspects are what drew us at iGEM Uppsala 2017 to work with the pathway from zeaxanthin to crocin in the BioBrick format, but also to integrate the metabolic steps in the pathway from farnesyl pyrophospate (FPP) to zeaxanthin on the chromosome of Escherichia Coli. The enzyme presented on this page, UGTCs2 catalyzes the second reaction in the zeaxanthin to crocin pathway.

In more detail UGTCs2 is a UDP-glucuronosyltransferase (UGT) that adds sugar ends to the carboxylic acid ends of crocetin forming crocin (3).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 758
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 931
  Illegal AgeI site found at 1286
  
• 1000
  COMPATIBLE WITH RFC[1000]

Modeling of UGTCs2

Since UGTCs2 lacked any type of structure we created an homology model to be able to determine where to put our his-tag. For the homology model we used 5v2k as a template (4) (figure 1). This model was then used in a 100 ns molecular dynamics simulation where the stability of the model of was evaluated. This yielded an RMSD plot (figure 1) and a movie (figure 2).

Figure 1. Homology model of UGTCs2 and RMSD plot for the same model. The simulation was run for 100 ns and displayed a stable homology model.

References

1. Papandreou MA, Kanakis CD, Polissiou MG, Efthimiopoulos S, Cordopatis P, Margarity M, et al. Inhibitory Activity on Amyloid-β Aggregation and Antioxidant Properties of Crocus sativus Stigmas Extract and Its Crocin Constituents. J Agric Food Chem. 2006 Nov 1;54(23):8762–8.

2. Chen L, Qi Y, Yang X. Neuroprotective effects of crocin against oxidative stress induced by ischemia/reperfusion injury in rat retina. Ophthalmic Res. 2015;54(3):157–68.

3. Moraga AR, Nohales PF, Pérez JAF, Gómez-Gómez L. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta. 2004 Oct 1;219(6):955–66.

4. George Thompson AM, Iancu CV, Neet KE, Dean JV, Choe J-Y. Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana. Sci Rep. 2017 Apr 20;7:46629.