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CaCCD2 with BBa_J04500

This BioBrick codes for the enzyme called CaCCD2 that is the first of three enzymes in the crocin pathway that starts from zeaxanthin. CaCCD2 catalyzes the reaction from zeaxanthin to crocetin dialdehyde. In nature the enzyme can be found in Crocus Sativus (the plant where saffron is harvested from). This BioBrick is sequence verified and we also created a homology model because of previously poor characterization. The homology model was used for a 100 ns molecular dynamics simulation, which concluded that the enzyme is stable.

This BioBrick contains a lac promoter which means that it has to be induced by lactose or some other closely related derivative, such as IPTG.

Previous iGEM teams have worked with another enzyme called ZCD that was thought to catalyze the same reaction, however no success has been seen with that enzyme.

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, CaCCD2 catalyzes the second reaction in the zeaxanthin to crocin pathway.

In more detail CaCCD2 is an carotenoid cleavage dioxygenase (CCD) that cuts zeaxanthin at 7,8 and 7',8' position forming crocetin dialdehyde and 3-OH-ꞵ-cyclocitral. This subclass enzymes (CCD2) was first mentioned in an article from 2014 (3) and one year later CaCCD2 was cloned and expressed (4).

Sequence and Features

Modeling

Since CaCCD2 lacks a structure we created an homology model of (see figure 1) it using the pdb entry 5j53 (5). This model was then used in a molecular dynamics simulation which ran for 100 ns to evaluate its stability (see figure 1 and 2).

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. Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado-Cabrero A, et al. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proceedings of the National Academy of Sciences. 2014 Aug 19;111(33):12246–51.

4. Ahrazem O, Rubio-Moraga A, Berman J, Capell T, Christou P, Zhu C, et al. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New Phytol. 2016 Jan 1;209(2):650–63.

5. McAndrew RP, Sathitsuksanoh N, Mbughuni MM, Heins RA, Pereira JH, George A, et al. Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase. Proc Natl Acad Sci U S A. 2016 Dec 13;113(50):14324–9.