Contribution
Overview
Our project harnessed the power of plant-derived UDP-glycosyltransferase (UGT). In the preliminary phase of our research, a comprehensive literature review revealed that UGTs are ubiquitous across various organisms, fulfilling crucial physiological functions. We have cataloged UGTs from diverse sources, laying a foundation for future research endeavors in this field. Additionally, our team made significant contributions to the development of an activity assay for UGTs. Reviewing past iGEM projects focusing on glycosyltransferases, we observed that the majority of activity assays relied on High-Performance Liquid Chromatography (HPLC), a method that is not only costly but also necessitates sophisticated instrumentation. Consequently, our literature review led to the identification of an alternative approach for assessing UGT activity, utilizing a microplate reader. Our experimental outcomes confirmed the suitability of this method for our enzyme of interest, Gt6CGT. In pursuit of establishing the versatility of this assay technique, we extended our investigation to include the activity measurement of two additional UGT. This expansion has yielded supplementary data, further validating the method's broad applicability.
UGTs classification
UDP-glycosyltransferases (UGTs) are an enzyme superfamily found in animals, plants, fungi, and bacteria. They catalyze the covalent addition of sugars from nucleotide UDP-sugar donors to functional groups (most commonly hydroxyl, carboxyl, or amine groups) on a wide range of lipophilic molecules [1]. UGTs are key phase II enzymes in detoxification systems that have evolved across all kingdoms of life [2]. Next, the UGTs from these different sources will be introduced.
1. Mammals
In mammals, the superfamily includes four families: UGT1, UGT2, UGT3, and UGT8. The members of these four families (22 in humans) are thought to have arisen through iterative gene duplication and divergence processes to perform biotransformation functions, or rarely, biosynthetic functions. Biotransformation plays a key role in helping eliminate countless potentially toxic exogenous chemicals, removing toxic byproducts of endogenous metabolism, and regulating the levels and distribution of endogenous signaling molecules [3]. For example, UGT3A1 and UGT3A2 are involved in conjugation with N-acetylglucosamine and glucose, respectively [4]; UGT8A1 participates in the conjugation of ceramide and bile acids with galactose, among others [5].
2. Plants
Plant-derived UGTs are mainly used in the biosynthesis of many terpene compounds, catalyzing the transfer of sugars from donors to acceptors, forming various bioactive glycosides [6]. Glycosylation is a key mechanism that determines the chemical complexity and diversity of plant natural products and influences their chemical properties and biological activities. Uridine diphosphate glycosyltransferases (UGTs) are central participants in these glycosylation processes, decorating natural products with sugars [7]. Glycosylation can enhance the solubility and stability of these natural products, promote their storage and accumulation in plant cells [8-10], and determine their bioactivity and bioavailability [11-12]. For example, flavonoids are a diverse group of plant natural products with over 7,000 known compounds [13]. They are usually present in glycosylated forms. Quercetin, a potent antioxidant, can be modified with various sugars at different positions, resulting in 300 different quercetin glycosides, each potentially having different biological activities. The attachment of rhamnose to the 2-OH or 6-OH of the glucose portion of naringin 7-O-glucoside determines the bitterness of grapefruit or the lack of taste in oranges [14]. Terpenoids are another large class of natural products, with more than 40,000 different compounds identified [15]. Saponins are terpenoid glycosides with antifungal properties, but the removal of their sugar residues often leads to a loss of bioactivity. The diterpene glycosides stevioside and rebaudioside A from Stevia leaves are extremely sweet compounds, and their different glycosylation patterns determine their taste perception [16]. In addition, plant UGTs also play important roles in the detoxification of xenobiotics and the regulation of plant hormone activity. For example, Arabidopsis UGT73C5 glucosylates the steroid hormone brassinosteroids, reducing their biological activity [17]. Arabidopsis UGT72B1 is involved in xenobiotic metabolism, such as the metabolism of pollutants 2,4,5-trichlorophenol and 3,4-dichloroaniline [18].
3. Insects
UDP-glycosyltransferase (UGT) is one of the major phase II detoxification enzymes in insects, involved in xenobiotic metabolism and potentially mediating the development of insect resistance [19]. UGT can detoxify toxic plant chemicals encountered in insect diets and also participates in various physiological processes, such as the formation of the cuticle, pigments, and olfactory senses. Insect UGT enzymes exhibit broad substrate specificity. Studies have shown that UGT enzymes in silkworms are active against substrates such as coumarins, terpenoids, flavonoids, phenolic compounds, and their derivatives. Experimental results have also demonstrated their crucial role in detoxification metabolism [20]. In addition, many UGTs are present in other organisms, such as fungi, bacteria and invertebrates, but the functions of these UGTs have not been well studied, so they will not be discussed in detail here.
Detection of UGTs activity from two different sources
From the above introduction, it is clear that UDP-glycosyltransferases (UGTs) from different sources play important roles in organisms, making the study of these enzymes' functions highly significant. In the research of gene function, enzyme activity assays are also a critical step. Traditional methods for detecting UGT activity include thin-layer chromatography (using radiolabeled co-substrates) or high-performance liquid chromatography (HPLC) [21, 22]. In both methods, one of the limiting factors in detecting total UGT activity is the selection of a general UGT substrate that works well for all UGT family members present in the sample. Other limiting factors include sample availability, cost, or substrate detection. Some substrates have good UV absorption properties, while others require fluorescence or mass spectrometry detection. These two methods are costly and require advanced equipment, making detection somewhat challenging. Therefore, through literature review, we identified a simpler method for UGT activity detection: using α-naphthol as the substrate and UDP-glucose as the glycosyl donor. The glycosylation reaction produces α-naphthyl glucoside, and the change in fluorescence intensity is monitored continuously at an excitation wavelength of 287 nm and an emission wavelength of 335 nm, with a slit width of 5 nm, to reflect the enzyme activity. Our experimental results show that this method is suitable for Gt6CGT. However, to further explore the broad applicability of this method, our team measured the activity of two other UDP-glycosyltransferases from different sources, adding new data to support this method.
1. Method
(1) Prepare the following system
(2) Add 10 μL of 10 μM α-naphthol and react at 37°C for 1 minute. Then, add the mixed solution to a black microplate at 200 μL per well, with three replicates per group. Use PB (phosphate buffer) as a control to replace the enzyme solution.
(3) Place the black microplate into the plate reader for detection, setting the following conditions: temperature at 37°C, excitation wavelength at 287 nm, emission wavelength at 335 nm, and slit width of 5 nm.
(4) Record the data and perform graph analysis.
2. Results
UGT1：derived from insects UGT2：derived from plants
Conclusion
From the results, it can be seen that both UGT1 and UGT2 exhibited detectable glycosyltransferase activity, indicating that this method is applicable to these two different UGT sources as well. Compared to previous UGT detection methods, this approach significantly reduces experimental costs and lowers the requirements for laboratory equipment. The experimental process is simple and easy to follow, providing a useful reference for future teams conducting UGT experiments.
References
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