Protocol for detecting threonine deaminase activity in fission yeast cell lysates

Summary Threonine deaminase catalyzes the first step of isoleucine biosynthesis from threonine. In this protocol, we describe the process of measuring the enzymatic activity of threonine deaminase in the fission yeast cell lysate, which is catalyzed by Tda1. First, we describe the process of preparing cell lysates from fission yeast cell cultures. Subsequently, we explain how to measure the threonine deaminase activity using threonine or serine as a substrate. For complete details on the use and execution of this protocol, please refer to Sasaki et al. (2022).1


SUMMARY
Threonine deaminase catalyzes the first step of isoleucine biosynthesis from threonine.In this protocol, we describe the process of measuring the enzymatic activity of threonine deaminase in the fission yeast cell lysate, which is catalyzed by Tda1.First, we describe the process of preparing cell lysates from fission yeast cell cultures.Subsequently, we explain how to measure the threonine deaminase activity using threonine or serine as a substrate.For complete details on the use and execution of this protocol, please refer to Sasaki et al. (2022). 1

BEFORE YOU BEGIN
This protocol was developed for measuring the threonine deaminase activity in fission yeast cell lysates.The extraction method is based on the previous report by McDonald and Kaplan in 1973, 2 while the color reaction for detecting the ketoacids which are the reaction products is based on the previous report by Datta in 1966 (Figure 1). 3 This protocol is potentially applicable for other organisms, fractions prepared from crude lysates, and purified proteins.b.Add a certain amount of the culture to 100 mL EMM2 in 300 mL flasks and cultivate for 24-48 h to reach 0.5 OD 595 .c. Shake the flasks at 125 rpm, at 27 C.

Note:
The doubling time is around 4 h and over, depending on the strains.
Note: When you want to cultivate cells in a media containing an amino acid as a sole nitrogen source, use the amino-acid supplemented media at steps 2 and 3.
CRITICAL: We strongly recommend starting the lysis process using the cell culture with OD 595 values of over 0.5 to ensure that the wet weight of collected cells is over 150 mg.
Next yeast cells are harvested and lysed.h.Remove the supernatant by aspiration.Weigh the tube to calculate the wet weight of the collected cells using a laboratory scale.You can store the yeast pellets: put the microtube in liquid nitrogen for a few seconds and store them in a -80 C deep freezer.Thaw before use.6.If the wet weight of cells is under 100 mg, add 100 mL lysis buffer to the microtube, and resuspend the cells.If the wet weight is over 100 mg and less than 160 mg, add 200 mL lysis buffer.
CRITICAL: Loading too many cells in one lysing tube prevents the cells from being completely crushed, resulting in low efficiency of protein extraction; do not dispense more than 160 mg wet cells in one lysing tube.
7. Put 0.5 g glass beads into a 2 mL lysing tube and add all the cell suspension prepared at step 6. 8. Homogenize cells 10 to 15 times for 30 s at 30 s interval using multi-beads shocker, depending on the amount of cell suspension.
Note: Observe the cell suspension under a light microscope to confirm that almost all cells are disrupted.After homogenization, be sure to always keep the lysates on ice.9. Add 100 mL lysis buffer to the lysing tube after homogenization and make a hole at the bottom of the lysing tube by using a push pin (Figure 2).Wipe off the homogenized solution from the push pin every time.10.Set the 2 mL lysing tube on a new 1.5 mL microtube (Figure 2), and centrifuge together for 2 min at 4 C, 20 3 g.11.Place the tubes on ice.Put your finger on the lysing tube to squeeze the extract out of the tube completely.12. Add lysis buffer to the 1.5 mL microtube so that the total volume will be 900 mL and centrifuge for 10 min at 4 C, 21,500 3 g.13.Transfer the supernatant to a new 1.5 mL microtube as a crude lysate.14.Measure the protein concentration of the crude lysate using Bradford method.a.To draw a standard curve, dilute 0.1% BSA using 0.084 M potassium phosphate buffer in 1.5 mL microtubes as shown below.The diluents can be prepared one at a time, or a stepwise dilution can be performed.0.1% BSA 0.084 M potassium phosphate buffer Total 0 mL 2 0 mL 2 0 mL 2 mL 1 8 mL 2 0 mL 4 mL 1 6 mL 2 0 mL 8 mL 1 2 mL 2 0 mL 16 mL 4 mL 2 0 mL b.Dilute 2 mL crude lysate with 18 mL 0.084 M potassium phosphate buffer in 1.5 mL microtubes as the samples for quantification.c.Add 1 mL Protein Assay Dye Reagent to the standard curve samples and lysate samples.d.Vortex for 5-10 s and allow to stand at 20 C-25 C for at least 5 min.e.Take 200 mL from the tubes and dispense them into a 96-well plate.f.Measure the OD 620 using a Multiskan FC plate reader.Be sure to measure the absorbance of the reacted protein solutions as soon as the process; otherwise, the products aggregate, and the experimenter will not be able to know the correct protein concentration of the cell lysates.g.Calculate the protein concentrations in samples using the standard curve.If the measured value of the lysate sample is larger than that of the standard samples, dilute 1 mL of crude lysate in 19 mL of 0.084 M potassium phosphate buffer, and repeat the procedure starting from step b.

Threonine deaminase assay
Timing: 1-1.5 h This step shows the way to measure the activity of threonine deaminase.
15. Prepare standard solutions.a. Dissolve a-ketobutyrate or pyruvate in RO water to obtain 1.0 mM solution and dilute it first with 0.17 M potassium phosphate buffer (pH 8.0) to half the concentration.b.Dilute the sample prepared at step a to half the concentration with 0.084 M potassium phosphate buffer (pH 8.0) five more times.
Note: When you use threonine as a substrate, use a-ketobutyrate; for serine, use pyruvate.
16. Add 10 mL of 30% TCA to each well in 96-well plates.17.Add 100 mL of standard samples or 0.084 M potassium phosphate buffer (pH 8.0) and 10 mL of 0.2% DNPH in 2 N HCl to wells with 30% TCA, which were prepared at step 16. 18. Prepare a substrate solution by dissolving threonine or serine in RO water.Mix the amino acid solution and pyridoxal phosphate solution.The final concentration of the pyridoxal phosphate is 20 mM, and the substrate concentration can be 5-40 mM (threonine) or 10-80 mM (serine).
CRITICAL: We found out that if amino acids are stored with pyridoxal phosphate for a long time, the product amount in the enzymatic assay decreased.It is better to prepare and store the solution independently and mix them just before the test.
19. Dilute lysate by 0.084 M potassium phosphate buffer (pH 8.0) in 1.5 mL tubes so that protein concentration will be 1 or 7 mg per 90 mL.Use the lysate with the protein concentration of 1 or 7 mg protein per 90 mL for testing threonine or serine as a substrate, respectively.

Note:
The volume of the mixture that should be prepared depends on the number of experiments.
20. Dispense 90 mL of the lysate to a 96-well plate containing 10 mL of 30% TCA, then dispense 10 mL solution containing amino acid and pyridoxal phosphate.This well will serve as a sample at t = 0. 21.Mix substrate and pyridoxal phosphate solution (10 mL) and the lysate (90 mL) in a microtube and incubate at 30 C. If you want to measure the activity at five time points, mix 60 mL of the substrate and pyridoxal phosphate solution and 540 mL of the lysate.22.After the specified time of experimenter's choice, in which the minimum time is 1 min, take 100 mL of the mixture from the microtube to a 96-well plate containing 10 mL of 30% TCA per well (prepared at step 16).
CRITICAL: Because enzyme reactions undergo every moment, make sure to stop the reaction using 30% TCA just at the moment when the reaction interval reaches the specified time.
23.After the reaction is stopped, add 10 mL of 0.2% DNPH in 2 N HCl in each well.Incubate the plate at 30 C for 10 min.24.Add 100 mL of 2.5 N NaOH to all the wells including standard samples and your cell lysate samples, and allow to stand at 20 C-25 C for 10 min.25.Measure the OD 540 values using a Multiskan FC plate reader.26.Calculate the amount of the reaction product in samples using the standard curve.
Note: If the measured value of the digested product is larger than the standard samples, reprepare the standard curve using higher concentrations of standard solution.
Optional: If you want to test enzyme inhibition or activation by amino acids, add isoleucine (around 0.1 mM) or valine (0.1-10 mM) to the substrate solution. 1

EXPECTED OUTCOMES
Threonine deaminase activity in the fission yeast cell lysates can be detected by following the steps described above.The enzymatic reaction products, a-ketoacids, can be detected using DNPH in 96 well plates by measuring OD 540 values (Figure 1).a-Ketobutyrate and pyruvate are generated from threonine and serine, respectively.You can obtain calculation curves by testing a series of concentrations of a-keto acids (Figures 3A and 3D).After measuring the TD activities in cell lysates (Figures 3B and 3E, left), you can calculate how much amount of a-ketobutyrate and pyruvate is produced using the calibration curve (Figures 3B and 3E, right).Concerning fission yeast Tda1, threonine is a more favored substrate than serine, so use more lysates when reacting serine than threonine as described in step 19.Reproducibility should be examined to confirm that your data is statistically meaningful (Figures 3C and 3F).You can examine various culture conditions and strains. 1 Results for wild-type and cells overexpressing tda1 are shown in Figure 3. Overexpression of tda1 resulted in increased enzymatic activity of the cell lysate, confirming that Tda1 is the enzyme responsible for the TD activity in this organism.

LIMITATIONS
This protocol can be used to test threonine deaminase activity in various fission yeast strains, and the obtained lysates can be reused if it is frozen rapidly in liquid nitrogen and stored in a deep freezer at -80 C.However, there are some limitations.Most importantly, the lysate obtained from this protocol is crude; purification using a protein tag, or any other method is necessary to exclude the effect of other metabolites and interacting proteins.At the same time, the protein tag at the C-terminus of Tda1 blocks its cellular function, 1 thus it is essential to express functional proteins with a tag sequence at its N-terminus.In addition, it is also important to keep in mind that the 1 or 7 mg total protein stated earlier to be used for Tda1 activity assay is not necessarily the amount of the Tda1 enzyme.

TROUBLESHOOTING Problem 1
The yeast culture does not reach an OD 595 of 0.5 in the flasks, at step 3.

Potential solution
Use freshly cultured yeast cells when preculturing in test tubes, instead of using cells that were stored in the refrigerator for a while.Changing the incubation time or rotation speed of the flask culture could be effective to shorten the doubling time.By the way, loading too many cells in one lysing tube prevents the cells from being completely crushed, resulting in low protein concentration; do not dispense more than 160 mg wet cells in one lysing tube.As stated in step 6, when the wet weight is more than 160 mg, it is suggested that the experimenter split the cells into multiple tubes.

Problem 2
The substrates are not deaminated enough to be calculated using the standard curve, at step 26.

Potential solution
First, the cells may not have been lysed enough, and consequently the Tda1 enzyme may not have been fully extracted.To prevent this, we strongly recommend conducting the cell-lysing process using the cell culture with OD 595 values of over 0.5 to ensure that the wet weight of collected cells measure over 150 mg.Also, be sure to always keep the lysates on ice to prevent the enzymes from deactivating.Another solution is to increase the amount of the cell lysate, elongate the reaction time, or prepare substrate solutions at higher concentration.In addition, one of the points that could be checked is how each solution is stored or prepared.We have found out that if substrates are stored with pyridoxal phosphate for a long time, the amount of the produced a-ketoacids decreases.It is better to prepare and store the solution independently and mix them just before the test.

Problem 3
The calculated amount of the product is not zero at 0 s of reaction, in which the sample is prepared at step 20 and measured at step 25.

Potential solution
The experimenter is probably following the protocol correctly.Our idea here is that a-keto acids such as a-ketobutyrate and pyruvate are contained in the crude cell lysate used in the enzymatic activity test, and they are detected by the coloring reagent.A secure solution would be to purify the Tda1 enzyme.Another idea is to use less lysate (less than 1 mg) and to elongate the reaction time to minimize the effect of the intracellular metabolites.

4.
Turn on the multi beads shocker.Set the temperature at 4 C to cool down the rotor.5. Harvest yeast cells.a.Weigh the 1.5 mL microtubes that will be used to collect yeast cells.b.Pour half of the culture into a 50 mL centrifuge tube, and centrifuge for 2 min at 4 C, 900 3 g.c.Remove the supernatant by decantation.Pour the rest of the culture into the same centrifuge tube, and centrifuge for 2 min at 4 C, 900 3 g.d.Remove the supernatant by decantation.e. Suspend the yeast pellet in 5 mL ice-cold sterilized RO water and centrifuge for 2 min at 4 C, 900 3 g.f.Remove the supernatant by decantation.g.Suspend the yeast pellet in 1 mL ice-cold sterilized RO water.Transfer the suspension into a 1.5 mL microtube that was weighed in advance (step a), and centrifuge for 1 min at 4 C, 2,400 3 g.

Figure 1 .
Figure 1.Reaction scheme of the threonine deaminase assay Amino acids are deaminated to produce a-ketoacids.a-Ketoacids are reacted with DNPH to be converted to 2,4-dinitrophenylhydrazones, which are detected by measuring the absorbance at 540 nm.

Figure 2 .
Figure 2. How to transfer cell lysates from lysing tubes (A) Make a hole at the bottom of the lysing tube by using a push pin.(B) Set the 2 mL lysing tube on a new 1.5 mL microtube.After centrifugation, the cell lysates can be collected in the bottom tube.

Figure 3 .
Figure 3. Examples of raw and calculated data of the TD activity Calibration curves for a-ketobutyrate (A) and pyruvate (D) are shown.a-Ketobutyrate that is enzymatically generated from threonine is detected by measuring OD 540 (B, left), whose amount is calculated using the calibration curve (B, right).Repeat more than three times, calculate and show the average and experimental error to yield the data (C).Data obtained for experiments using serine as a substrate are shown in (E) and (F).Wild-type cells (black) and cells overexpressing tda1 (red) were cultivated in the modified EMM2 medium in which serine (5 mM) was used as a sole nitrogen source. 1 Data in (C) and (F) represent the mean G SD (n = 3).Graph shown in (C) and (F) are modified and reproduced from Sasaki et al., 2022. 1 1. Prepare 40% glucose solution and autoclave it at 121 C for 20 min..Add autoclaved glucose solution and filter-sterilized salt stock to the autoclaved EMM2 without glucose and salt stock.e.You can replace NH 4 Cl with amino acids as nitrogen sources (# in the EMM2 recipe).1Aminoacid was added to the minimal media and autoclaved for sterilization, before glucose solution and salt stock were added.2. Preparation of the solid YES medium.Inoculate fission yeast cells from YES plates into 5 mL EMM2.Cultivate for 16-24 h at 27 C in test tubes, which are rotated at around 40 rpm.3.Cultivate fission yeast cells in a larger scale.a.Measure the OD 595 value of the yeast culture of step 2.
i. 40% glucose: add 40 g glucose in RO water, fill up to 100 mL.c.Prepare EMM2 without glucose solution and salt stock (* in the table below), and autoclave it at 121 C for 20 min.da.Prepare the solid YES medium and autoclave it at 121 C for 20 min.Filtrate using a 0.22 mm filter.Prepare 15 mL aliquots.Store at -20 C for several months, or at 4 C for some months after thawing the aliquot.(Continued on next page) MATERIALS AND EQUIPMENT 30% trichloroacetic acid: add 15 g trichloroacetic acid in RO water, fill up to 50 mL.2.5 N NaOH: add 5 g NaOH in RO water and fill up to 50 mL.400mMpyridoxalphosphate:add1.6 mg pyridoxal phosphate monohydrate in RO water and fill up to 15 mL.Keep this solution at 4 C. *Add DTT (1/500 volume of 1 M solution; final concentration, 2 mM) and PMSF (1/100 volume of 100 mM solution; final concentration, 1 mM) just before use.Store at 4 C for some months.Cover the container with aluminum foil for shading.STEP-BY-STEP METHOD DETAILSObtaining crude cell lysate from fission yeast cells Timing: 3-5 daysThis step explains how yeast cells are cultivated.1.Streak out fission yeast strains on solid YES media and incubate for three to five days at 27 C, depending on the strain.Fission yeast strains are stocked in 20% glycerol (v/v) at -80 C. 2.