Analysis of ergothioneine biosynthetic enzymes Stiftung Schweizer Jugend forscht Lea Egli, Gymnasium Köniz-­‐Lerbermatt Supervisor: Laëtitia Misson, Universität Basel Introduction: Ergothioneine is a vitamin, which is found in plants, fungi, bacteria and also in the human body. Its biosynthesis has been identified in 20101. However, its exact function in our body remains unknown. My supervisor works in a research group, which aims at understanding ergothioneine function in our body. The first step of ergothioneine biosynthesis is the N -­‐trimethylation of histidine and it’s catalysed by an enzyme called EgtD. EgtD is a S-­‐adenosylmethionine (SAM) dependant enzyme. SAM is the donor of the methyl group in the reaction. Figure 1 Ergothioneine biosynthesis in mycobacteria My project consisted in measuring the rate of the reaction of the methylation of histidine. To do so, I used a “trick”. Two additional enzymes were added in the reaction in order to follow the reaction by using the corresponding change in absorption2. I was then able to determine different rates of histidine methylation catalysed by four different enzymes. Astract: Ergothioneine is a vitamine found in plants, fungi and bacteria. This vitamin is not produced in our body, but we take it from the food. We still don’t know what is the exact role of ergothioneine in our body, even if its biosynthesis in bacteria has been identified1. In my project I worked on the first step of ergothioneine biosynthesis which is the methylation of histidine catalysed by EgtD in presence of SAM (figure 1). Laëtitia Misson studied the crystal structure of EgtD. She identified important amino acids in EgtD, which may be important for the reaction. To test this hypothesis, she did three different mutations on EgtD WT. We measured the rate of the reactions catalysed by four different enzymes: the wild type form of EgtD and three mutants A, B and C. We determined the rate of the reactions with the spectrophotometer. In order to use this instrument, we had to use two more enzymes, to be able to see a change in the absorbance at a specific wavelength. 1 Seebeck, F.P., 2010, J. Am. Chem. Soc., 132, 6632–6633 2 Dorgan, K.M. et. al., 2006, Anal. Biochem. 350, 249–255 We measured four different rates using the change in absorbance occurring during the four reactions. The three mutants seemed to be slower than EgtD WT. So the mutations changed the activity of the enzyme. The next step will be to determine why the reactions catalysed by mutants are slower, that is to answer the following question: which roles play the mutated amino acids in the catalysis. Material & Methods: We prepared a 0.2 mmol/L solution of histidine, the enzymes and SAM solutions were already made. Then we mixed our compounds according to the following tables. Table 1 Initial and final concentrations of the different compounds of the reactions stock solution Volume in the cuvette concentration in the cuvette EgtD (wild type) Histidine SAM Enzym 1 Enzym 2 37 µmol/L 10 mmol/L 25 mmol/L 370 µmol/L 140 µmol/L 6.8μL 20μL 8μL 6.8μL 8.9μL 0.25μmol/L 0.2mmol/L 0.2mmol/L 2.5μmol/L 1.25μmol/L Table 2 Pipetting scheme of the reactions (Vtotal = 1 000 µL) Reaction Buffer Histidine SAM Enzyme 1 Enzyme 2 Methyltransferase 1 2 3 4 949.5µmoL 946.1µmoL 946.9µmoL 944.5µmoL 20µL 20µL 20µL 20µL 8µL 8µL 8µL 8µL 6.8µL 6.8µL 6.8µL 6.8µL 8.9µL 8.9µL 8.9µL 8.9µL EgtD WT:6.8 μL Mutant A:10.2 μL Mutant B:9.4 μL Mutant C:11.8 μL We mixed first the buffer (Tris pH8 50 mmol/L, NaCl 50 mmol/L, Mn2+ 100 µmol/L) with histidine, with SAM and with the enzymes 1 and 2 in the cuvette. The enzyme 1 is nucleosidase and the enzyme 2 is adenine deaminase. Their respective roles are shown on figure 2. We measured the absorbance of this solution in the spectrophotometer. No change in absorbance was observed. Then we added the methyltransferase (either EgtD wild type, or mutant A, or B or C) and mixed well. We put the cuvette back in the spectrophotometer and measured the absorbance. We could then observe a change in absorbance. With each methyltransferase we did two measurements. Results: We measured different decreases of absorbance at 265nm for each methyltransferase (EgtD WT, mutant A, B or C). The slopes are indicated in table 3. The decrease of absorbance only happened as the methyltransferase was added to the solution. The figure 3 shows how the linear decrease in absorbance looks like. When we did the reaction with the EgtD WT we could see a decrease of the absorbance after the addition of EgtD WT. When we did the reaction with the mutants, the decreases in absorbance happened, but they were not so strong. To convert the change in absorbance into a change in concentration, the rate of the reaction, we use the Beer-­‐Lambert law. As the extinction coefficient of the reaction at 265nm is 6 700 mol-­‐1 L cm-­‐1, we were able to calculate the corresponding rates. An example is shown below: EgtD WT: dAbs/min = 0.0194 Beer-­‐Lambert law: A = (mol-­‐1 L cm-­‐1) x l (cm) x c (mol/L). We know that l = 1cm and = 6 700 mol-­‐1 L cm-­‐1. Then: 0.0194 (dAbs/min)= 6 700 (mol-­‐1 L cm-­‐1) x 1(cm) x c(mol/L/min) c (mol/L/min) = 0.0194(dAbs/min)/[ 6 700 (mol-­‐1 L cm-­‐1) x 1(cm)] c= 2.895 x10-­‐6 mol/L/min. The other rates are shown in the following table. Table 3 Rates of histidine methylation catalyzed by the 4 different enzymes rate of the reaction in mol/L/min Methyltransferase decrease in absorbance in Abs/min EgtD WT Mutant A Mutant B Mutant C Measurement 1 -­‐0.0196 -­‐0.001 -­‐0.0005 -­‐0.0008 Measurement 2 -­‐0.0192 -­‐0.0009 -­‐0.0007 -­‐0.0008 |Average| 0.0194 0.00095 0.0006 0.0008 2.895 x 10-­‐6 1.418 x 10-­‐7 8.955 x 10-­‐8 1.194 x 10-­‐7 Discussion: In our experiments, we could see that the rates of the reactions catalysed by the mutants are at least 20 times slower than the one catalysed by EgtD WT. The decreases in the absorbance were visible because of a trick (figure 2), which is the use of two additional enzymes. Figure 2 Scheme of the reactions leading to the decrease in absorbance at 265 nm When SAM, the donor of the methyl group gives its methyl group to histidine, the enzyme 1 can cut it into two pieces. One of these pieces is adenine. Now the enzyme 2 changes the NH2 group of adenine into an oxygen and that’s what we see as decrease in the absorbance at 265nm. If this reaction is fast, the slope is steep. But if the reaction doesn’t occur, SAM keeps its methyl group and we see no decrease in absorbance When we did the reactions with the mutants, we observed almost a flat line, whereas the slope was really steep with EgtD WT. The reactions with the mutants are very slow. That means that Laëtitia Misson may have changed an amino acid which is important for this reaction. To find out which role might have the mutated amino acids for the catalysis, supplementary experiments will be carried out. O n ly ad en in e
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Figure 3 Decrease in absorbance at 265 nm due to the deamination of adenine EgtD mutant A EgtD mutant B EgtD mutant C Figure 4 Decrease of the Absorbance at 265nm when the mutants are used (* indicates the moment when the methyltransferase is added). Acknowledgement: First I would like to thank the Stiftung Schweizer Jugend forscht, for this opportunity to spend a week in a research lab. I’m very happy that I had this great chance to go to this study week. I would also like to thank a lot the University of Basel, which gave me this place for studying in a lab. And my last thank goes to my supervisor Laëtitia Misson, who really made me understand the experiments that we carried on and never gave up when I didn’t catch the meaning in the first try. Literature: Seebeck, F.P., 2010, J. Am. Chem. Soc., 132, 6632–6633 Dorgan, K.M. et. al., 2006, Anal. Biochem. 350, 249–255