The 2018 UNSW Sydney iGEM team is proposing to synthesise a molecular scaffold for joining together disparate proteins in order to speed up the multi-step enzymatic reactions involved in the Indole acetic acid pathway. This pathway is important as Indole acetic acid is the most common and most studied plant hormone of the auxin class (promotes cell elongation).

 

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The UNSW Sydney 2018 iGEM team.

The concept is based on the use of Prefoldin to create a protein scaffold upon which we will attach different enzymes, using a parts-based system. A common prefoldin scaffold will be synthesised, to which many enzymes can be easily attached via the expression of particular, non-cross-reactive tags on the enzymes that will correspond to catchers on the protein (catchers and tags are small peptides that can be used to covalently join different proteins in a modular fashion). This structure will greatly reduce the diffusion distance of reaction components involved in multi-step enzymatic reactions, and therefore increase reaction rates and lead to greater efficiency of product creation.

Attaching an enzyme system to a protein scaffold is beneficial for enzymatic pathways as it allows for amplification of the effective concentration of each constituent reaction, enhances the stoichiometry of the enzyme system and averts the loss of substrates to alternative pathways. Furthermore, there is a decrease in waste products as well as process costs. Non-scaffolded enzyme systems have comparatively lower yields and are a costlier process. A protein scaffold brings the substrate close to enzyme 100% of the time, leading to an increase in the yield of the reaction product and favourable diffusion kinetics. Hence, UNSW iGEM wants to take advantage of scaffolded enzyme systems in order to produce a modular scaffold to which different multi-step enzyme systems can be attached.

The protein scaffold that the iGEM team is synthesising is the Prefoldin hexamer. This complex is made up of two classes of sub-units, α and β, that are evolutionary-related, and have been isolated from Methanobacterium thermoautotrophicum. The hexamer itself has two α and four β sub-units. Prefoldin is a molecular chaperone protein that facilitates the covalent folding or unfolding of other macromolecular structures.

The enzyme pathway the UNSW iGEM team is incorporating into the protein scaffold is part of the Indole acetic acid (IAA) biosynthesis pathway. IAA is a plant growth hormone (Auxin) and plays a role in plant-microbe relations. Auxins help in the maturation of the plant during fruiting, flowering and root initiation. Therefore, improvements in the IAA synthesis pathway are of interest to the agricultural industry. UNSW iGEM are using this pathway as it is a simple two-step reaction which can serve as a proof of concept to show that our Prefoldin scaffold did in fact increase the rate of a multi-step enzymatic reaction. The enzymes involved in this reaction are the tryptophan 2-monooxygenase (IaaM) (this is the rate limiting step of the multi-step reaction) and Indole-3-Acetamide Hydrolase (IaaH).

 

The reaction:

Tryptophan → IaaM enzyme → Indole acetamide → IaaH enzyme → Indole acetic acid

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E1: IaaH, E2: IaaM. Image credit: Brian Ee, 2018 UNSW iGEM team member.

The colocalization of the enzymes (IaaM and IaaH) together on a scaffold will reduce the distance required for the intermediate products of the reaction to diffuse and reach the next enzyme, increasing the reaction rate and yield of Indole acetic acid

What is exciting about the protein scaffold is that it will be a modular system. The enzymes in the Indole acetic acid pathway can be de-attached and other enzymes involved in different industries can be added, with the scaffold increasing the rate of reaction of many multi-step enzymatic pathways. This increases the potential of the prefoldin hexamer scaffold to be used in pharmaceutical industries, laundry detergents and fermented food and beverages.

 

Written by Zeeshan Siddiqui

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