A Novel Assay Method to Determine the Β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1

antioxidants

Article A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1

Arun Kumar Selvam and Mikael Björnstedt *

Department of Laboratory Medicine, Division of Pathology F46, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; [email protected] * Correspondence: [email protected]; Tel.: +46-085-858-3809

Received: 30 December 2019; Accepted: 3 February 2020; Published: 5 February 2020

Abstract: Kynurenine aminotransferase 1 (KYAT1 or CCBL1) plays a major role in Se-methylselenocysteine (MSC) metabolism. It is a bi-functional enzyme that catalyzes transamination and beta-elimination activity with a single substrate. KYAT1 produces methylselenol (CH3SeH) via β-elimination activities with MSC as a substrate. This methylated selenium compound is a major cytotoxic selenium metabolite, causing apoptosis in a wide variety of cancer cells. Methylselenol is volatile and possesses extraordinary nucleophilic properties. We herein describe a simple spectrophotometric assay by combining KYAT1 and thioredoxin reductase (TrxR) to detect CH3SeH in a coupled activity assay. The metabolite methylselenol and its oxidized form from MSC metabolism is utilized as a substrate for TrxR1 and this can be monitored spectroscopically at 340 nm. Our results show the feasibility of monitoring the β-elimination of KYAT1 by our assay and the results were compared to the previously described β-elimination assays measuring pyruvate. By using known inhibitors of KYAT1 and TrxR1, we further validated the respective reaction. Our data provide a simple but accurate method to determine the β-elimination activity of KYAT1, which is of importance for mechanistic studies of a highly interesting selenium compound.

Keywords: kynurenine aminotransferase 1; Se-methylselenocysteine; β-elimination activity; methylselenol; thioredoxin reductase; KYAT1 inducers; and KYAT1 inhibitors

1. Introduction Se-methylselenocysteine (MSC) is an organic methylated selenium compound that has drawn the attention of pharmacologists because of its high bioavailability, favorable pharmacokinetics and pronounced tumor-speciﬁc cytotoxic activity by altering numerous cell signaling pathways [1]. MSC itself is not an active drug, but the metabolites formed by enzymatic transformation have been reported to possess cancer prevention and anti-neoplastic properties [2]. Kynurenine aminotransferase 1 (KYAT1) plays a major role in MSC metabolism. KYAT1 is a multifunctional PLP-dependent enzyme involved in cleaving a carbon–sulfur bond, which transforms the amino acids into alpha-keto acids. This enzyme has dual activity—a transamination activity, and a beta-elimination activity. KYAT1 is mostly found in the cytosol. Se-conjugate compounds have been shown to favor beta-elimination activity over corresponding S-conjugate compounds. This may be due to a weaker C–Se bond than the C–S bond [3,4]. KYAT1 utilizes Se-methylselenocysteine (MSC) as a substrate to produce methylselenol (MS), pyruvate, and ammonium via β-elimination [5,6] and β-methylselenopyruvate (MSP) via transamination [2,7], as shown in Figure1. Both metabolites of MSC by KYAT1, i.e., MS and MSP, possess anti-tumor and anti-proliferative properties [2,8–10]. Of these, methylselenol (MS) is considered an important selenium metabolite because of its selective cytotoxic eﬀects towards tumor cells [2,11,12]. Because of this, it is necessary to measure the activity of KYAT1 when MSC is used as a substrate and to determine

Antioxidants 2020, 9, 139; doi:10.3390/antiox9020139 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 139 2 of 14 Antioxidants 2020, 9, 139 2 of 15 theof relation KYAT1 between when MSC transamination is used as a substrate and β-elimination. and to determine Considering the relation the greatbetween potential transamination of MSC as a chemotherapeutic,and β‐elimination. anConsidering accurate andthe great simple potential assay method of MSC to as measure a chemotherapeutic, the β-elimination an accurate activities and in varioussimple tumor assay samples method is to of measure great value. the β‐ Thus,elimination the β-elimination activities in activity various of tumor KYAT1 samples could beis aof valuable great biomarkervalue. Thus, for determining the β‐elimination which activity tumor of would KYAT1 respond could to be MSC a valuable treatment. biomarker Methylselenol for determining eﬃciently redoxwhich cycles tumor with would mammalian respond thioredoxin to MSC reductasestreatment. (TrxR1)Methylselenol [13]. Thioredoxin efficiently reductase redox cycles (TrxR, with TR) is anmammalian important enzyme thioredoxin in maintaining reductases the (TrxR1) redox [13]. homeostasis Thioredoxin by regulating reductase the(TrxR, redox TR) state is an of important thioredoxin, a wideenzyme range in maintaining of protein, lowthe redox molecular homeostasis weight by thiols, regulating and redox-active the redox state compounds. of thioredoxin, It is one a wide of the majorrange antioxidant of protein, systems low molecular in the cells weight [14 ].thiols, TrxR and reduces redox disulﬁdes‐active compounds. in the cell byIt is Trx one and of NADPH.the major antioxidant systems in the cells [14]. TrxR reduces disulfides in the cell by Trx and NADPH.

Figure 1. Schematic representation of β-elimination and transamination activity of the kynurenine Figure 1. Schematic representation of β‐elimination and transamination activity of the kynurenine aminotransferase 1 (KYAT1) enzyme. aminotransferase 1 (KYAT1) enzyme. There are many simple spectrophotometric protocols available to determine the transamination There are many simple spectrophotometric protocols available to determine the transamination activity of KYAT1 with various substrates, but there are only limited published possibilities to measure activity of KYAT1 with various substrates, but there are only limited published possibilities to the β-elimination activity, because of its limited substrate availability and complex assay procedure. measure the β‐elimination activity, because of its limited substrate availability and complex assay β Detectingprocedure. the Detecting-elimination the β‐ productelimination from product MSC is from difficult MSC because is difficult methylselenol because methylselenol is highly volatile is highly and highlyvolatile nucleophilic and highly [8]. nucleophilic There exist few[8]. There assays exist available few assays to measure available pyruvate, to measure which pyruvate, is one of the which products is of theoneβ of-elimination the products activity of the of β‐ KYAT1elimination from activity MSC and of itKYAT1 is determined from MSC by and the 2, it 4-dinitrophenylhydrazineis determined by the 2, (DNPH)4‐dinitrophenylhydrazine method [5,6]. However, (DNPH) this method is an indirect [5,6]. measurementHowever, this ofis methylselenol,an indirect measurement which measures of themethylselenol, amount of pyruvate, which measures i.e., it is the assumed amount but of not pyruvate, proven i.e., that it theis assumed amount but of pyruvate not proven generated that the is equalamount to the of methylselenol pyruvate generated (1:1 ratio) is equal generated to the [ 8methylselenol]. Thus, a major (1:1 problem ratio) generated in this assay [8]. is Thus, the uncertainty a major asproblem to what in is reallythis assay measured. is the uncertainty Furthermore, as to the what DNPH is really method measured. is unable Furthermore, to distinguish the DNPH between pyruvatemethod and is otherunable 2-oxo-acids to distinguish [9]. This between clearly pyruvate demonstrates and theother need 2‐oxo for‐ improvedacids [9]. assayThis methods.clearly Indemonstrates this study, we the developed need for a improved modified βassay-elimination methods. assay In this [8] bystudy, coupling we developed KYAT1 β-elimination a modified β‐ with TrxRelimination mediated assay reduction. [8] by coupling KYAT1 β‐elimination with TrxR mediated reduction. TheThe coupled coupled activity activity assay assay/TrxR1/TrxR1 was verifiedwas verified with KYAT1with KYAT1 transamination transamination assay and assay a preexisting and a β-eliminationpreexisting β‐ assayelimination/pyruvate. assay/pyruvate. Furthermore, Furthermore, we examined we a examined few compounds a few compounds that could that be potential could candidatesbe potential as inducers candidates or inhibitorsas inducers of or KYAT1 inhibitors transamination of KYAT1 andtransamination/or β-elimination and/or activity. β‐elimination We have furtheractivity. validated We have our further coupled validated activity assayour coupled data using activity these assay inhibitors data andusing inducers. these inhibitors and inducers. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals and Reagents 2.1. Chemicals and Reagents Potassium dihydrogen phosphate, di-potassium hydrogen phosphate, EDTA, sodium hydroxide, aminooxyaceticPotassium acid dihydrogen (AOAA), l-tryptophanphosphate, di (l‐-Try),potassium indole-3-pyruvic hydrogen acidphosphate, (IPA), phenylpyruvicEDTA, sodium acid (PPA),hydroxide,α-Keto- aminooxyaceticγ-methylthiobutyric acid acid(AOAA), sodium L‐tryptophan salt (KMB), ( se-methylselenocysteineL‐Try), indole‐3‐pyruvic hydrochlorideacid (IPA), phenylpyruvic acid (PPA), α‐Keto‐γ‐methylthiobutyric acid sodium salt (KMB), se‐ (MSC), dimethyl-2-oxoglutarate (α-KG), l-phenyl alanine (l-Phe), 2-amino-2-methyl-1,3-propanediol, methylselenocysteine hydrochloride (MSC), dimethyl‐2‐oxoglutarate (α‐KG), L‐phenyl alanine (L‐ pyridoxal 5 -phosphate hydrate (PLP), 2,4-dinitrophenylhydrazine (DNPH), Phenylmethanesulfonyl Phe), 20‐amino‐2‐methyl‐1,3‐propanediol, pyridoxal 5′‐phosphate hydrate (PLP), 2,4‐ fluoride (PMSF), RIPA buffer, protease inhibitor cocktail mix, and N-N-dimethyl formamide, pLKO.1 dinitrophenylhydrazine (DNPH), Phenylmethanesulfonyl fluoride (PMSF), RIPA buffer, protease vector were purchased from Sigma-Aldrich (Darmstadt, Germany). BFF-122 was purchased from Axon inhibitor cocktail mix, and N‐N‐dimethyl formamide, pLKO.1 vector were purchased from Sigma‐ MedchemAldrich (Groningen,(Darmstadt, Netherlands),Germany). BFF NADPH‐122 was was purchased purchased from from AcrosAxon OrganicsMedchem (New (Groningen, Jersey, NJ, USA).Netherlands), Plasmid pEGFP-N1 NADPH was (Clontech, purchased Takara from BioAcros USA, Organics Inc, Mountain (New Jersey, View, NJ, CA, USA). USA) Plasmid was a pEGFP generous‐ giftN1 from (Clontech, Dr. Gildert Takara Lauter Bio USA, from Inc, the Mountain Department View, for BiosciencesCA, USA) was and a generous Nutrition, gift Karolinska from Dr. Institutet,Gildert Stockholm,Lauter from Sweden. the Department HEPG2 cells for Biosciences and EMEM and media Nutrition, were Karolinska purchased Institutet, from ATCC Stockholm, (Wesel, Germany). Sweden. HEPG2 cells and EMEM media were purchased from ATCC (Wesel, Germany). Antioxidants 2020, 9, 139 3 of 14

2.2. Cell Culture The HEPG2 cell line was purchased from ATCC (Wesel, Germany) and maintained in EMEM (Eagle’s Minimum Essential Medium, ATCC) medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Gibco, Paisley, UK) under 5% CO2 at 37 ◦C without the addition of antibiotics.

2.3. KYAT1 Over-Expression The full-length KYAT1 coding sequence was amplified from cDNA and subsequently cloned into the pEGFP-N1 (Clontech, TakaraTakara Bio USA, Inc) mammalian expression vector. The human PGK promoter and Puromycin resistance gene sequences were amplified from pLKO.1 (Sigma Aldrich, Darmstadt, Germany ) and inserted into pEGFP-N1 backbone containing the KYAT1 expression vector. This KYAT1 over-expression plasmid was created for another ongoing study. An empty vector was created by inserting the PCR-amplified puromycin resistance gene expression cassette into pEGFP-N1 directly (manuscript in preparation). HEPG2 cells were transfected with either the KYAT1 over-expression plasmid or the empty vector using Lipofectamine 3000 (Invitrogen, Camarillo, CA, USA) according to the manufacturer’s protocols. Briefly, cells were seeded in 60-mm dishes 24 h before transfection. The cells were 75–90% confluent at the time of transfection. Then, 2 µg plasmid was mixed with 6 µL Lipofectamine 3000 and 5 µL P3000 reagent in a total volume of 500 µL OptiMEM medium (Gibco, ) and incubated for 10–15 min at room temperature before the transfection mixture was added dropwise to the cells. The cells were harvested after 48 h of transfection and subjected to protein isolation as described in Section 2.4.

2.4. Determination of Protein Concentration Samples were lysed in ice for 30 min in RIPA buﬀer (Sigma, Darmstadt, Germany) in the presence of 1 mM PMSF and 1% Protease inhibitor cocktail mix (Sigma). Further, lysed cells were sonicated at 4 ◦C for 30 s with 1- to 2-s pulses. The proteins were harvested by centrifuging at 13,000 rpm for 10 min at 4 ◦C and the supernatant was collected, and protein concentration was determined by the Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fischer Scientiﬁc, Rockford, IL, USA) according to the manufacturer’s protocols.

2.5. Enzymes KYAT1: Recombinant protein of KYAT1 (228 ng/µL), transcript variant 1 was purchased from OriGene Technologies (Rockville, MD, USA, Cat no: TP316317, Lot no: 111512). TrxR1: Mammalian TrxR1 was purchased from IMCO (Stockholm, Sweden) and Sigma-Aldrich (Cas no:9074-14-0, Product number T9698, Darmstadt, Germany).

2.6. Buﬀer Preparation

100 mM potassium phosphate buffer contained 80.2 mL of 100 mM K2HPO4, 19.8 mL of 100 mM KH2PO4 and 2 mM of EDTA, and the pH was adjusted to 7.4 with 1 M NaOH. The ammediol-HCL buffer contained 50 mL of 200 mM 2-amino-2-methyl-1,3-propanediol (21.03 g/L) and the pH was adjusted to 9.0 with 200 mM HCl (approximately 16.7 mL) and distilled water was added to a final volume of 100 mL. Preparation of 10 mM DNPH; 39.6 mg of DNPH was dissolved in 10 mL of 2 M HCl.

2.7. Enzyme Assay

2.7.1. Transamination Assay KYAT1 transamination activity was measured with pure protein or whole-cell lysate according to the published procedure [15,16]. Briefly, 50 µL of the reaction mixture contained 20 mM of l-Phe, 5 mM of KMB, 100 mM of ammediol-HCl buffer (pH 9.0) and 50 ng to 400 ng of pure KYAT1 protein or 20 µg of Antioxidants 2020, 9, 139 4 of 14

protein from whole-cell lysate. The assay mixture was incubated at 37◦C and the reaction was terminated by adding 150 µL of 1 M NaOH at different time points. The production of phenylpyruvate-enol was assessed by measuring the absorbance at 320 nm in a spectrophotometer (PowerWave HT, BioTek, Winooski, VT, USA). The assay mixture without enzyme was used as blank. Under this condition, 1 1 the molar extinction coefficient is 16,000 M− cm− for phenylpyruvate-enol [15]. UV flat-bottom plates (Corning, Kennebunk, ME, USA) were used for this assay. To assess the effect of transamination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 50 ng of KYAT1 enzyme. The reaction was pre-incubated for 5 min at 37 ◦C before being added the enzyme and KMB.

2.7.2. Beta-Elimination Assay/Pyruvate KYAT1 β-elimination activity was measured with pure protein or whole-cell extract according to the published procedure [17]. The assay protocol was adjusted to measure pure protein activity in 96-well plate readers for users’ convenience. Briefly, 50 µL of the reaction mixture contained 100 mM potassium phosphate buffer pH 7.4, 5 mM of MSC, 100 µM of αKG, 100 µM of KMB, 10 µM of PLP and 25 ng to 400 ng of pure KYAT1 protein or 20 µg of protein from whole-cell lysate. The reaction mixture was incubated at 37 ◦C and the reaction was terminated by adding 20 µL of 10 mM 2,4-dinitrophenylhydrazine in 2 M HCl at different time points. Then, 70 µL of this assay mixture was incubated at 37 ◦C for 10 min, 130 µL of 1 M NaOH was added and increases in absorbance were measured at 515 nm [18] in a spectrophotometer (PowerWave HT, BioTek). The assay mixture without 1 1 enzyme was used as blank. Under this condition, the molar extinction coefficient is 16,000 M− cm− for pyruvate 2, 4-dinitrophenylhydrazone. The reaction was pre-incubated for 5 min at 37 ◦C before adding the enzyme. To assess the effect of β-elimination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 200 ng of KYAT1 enzyme.

2.7.3. Coupled Activity Assay/TrxR1 Our coupled activity assay combines 2 different enzyme activities to detect methylselenol production, i.e., thioredoxin reductase 1 and KYAT1. Briefly, 100 µL of reaction mixture contained 100 mM potassium phosphate buffer pH 7.4, 1–10 mM of MSC, 100 µM of αKG, 100 µM of KMB, 10 µM of PLP, 0.4 µg mammalian TrxR1, 400 µM of NADPH and 50 ng to 400 ng of pure KYAT1 protein or 20 µg of protein from whole cell lysate. The reaction mixture was pre-incubated for 5 min at 37 ◦C before adding TrxR1 and NADPH. Continuous measurement of NADPH consumption was recorded at 340 nm for every 30 s using a spectrophotometer (PowerWave HT, BioTek). The assay mixture without TrxR1 was used as 1 1 blank. Under this condition, the molar extinction coefficient was 6220 M− cm− [19,20] for NADPH. Tmax represents the time taken to consume all the available NADPH in the assay mixture. To assess the inhibitory effects of β-elimination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 100 ng of KYAT1 enzyme.

2.8. Statistical Analysis The results are expressed in Box and Whisker plots showing median, 25- and 75- percentiles. The analysis was performed by either student t-test or one-way ANOVA with 95% conﬁdential interval followed by Dunnett multiple comparison tests (* p < 0.05, ** p < 0.01, *** p < 0.01 and **** p < 0.0001) compared to control. The data were analyzed with GraphPad Prism software, version 6 (GraphPad Software Inc, San Diego, CA, USA).

3. Results

3.1. TrxR1 and KYAT1 Coupled Enzyme Reaction The β-elimination activity of KYAT1 with MSC as a substrate was shown by Ioanna Andreadou et al. [8] using HPLC, which measured the amount of pyruvate formed. Later, J.L. Cooper et al. [15] used the DNPH Antioxidants 2020, 9, 139 5 of 15

3. Results

3.1. TrxR1 and KYAT1 Coupled Enzyme Reaction The β‐elimination activity of KYAT1 with MSC as a substrate was shown by Ioanna Andreadou et al. [8] using HPLC, which measured the amount of pyruvate formed. Later, J.L. Cooper et al. [15] used the DNPH method to quantify pyruvate. In this study, we modified the J.L. Cooper et al. [15] protocol and added TrxR1 into the assay system. We could observe the consumption of NADPH by a decrease in absorbance at 340nm (Figure 2a). This indicated that TrxR1 utilizes the product of the β‐elimination activity of KYAT1 (methylselenol) as its substrate, resulting in redox cycles and NADPH consumption [21]. AntioxidantsFigure2020 2b, shows9, 139 TrxR1 activity over time in this coupled activity assay. The following reaction5 of 14 is nonstoichiometric if there is access to oxygen and NADPH, i.e., most of the NADPH was consumed methodwithin 30 to min. quantify The consumption pyruvate. In of this NADPH study, weincreased modified with the increased J.L. Cooper substrate et al. [ 15concentration.] protocol and The added maximum time taken to consume all the NADPH was >120, 83.6, 53.6, 35.0, 27.0 and 24.6 min for TrxR1 into the assay system. We could observe the consumption of NADPH by a decrease in absorbance at 1,2,4,6,8 and 10 mM of MSC, respectively (Figure 2c), and Figure 2d shows the quick clearance of 340 nm (Figure2a). This indicated that TrxR1 utilizes the product of the β-elimination activity of KYAT1 NADPH, i.e., there is only a little NADPH available after 45 min, so it is necessary to calculate the (methylselenol) as its substrate, resulting in redox cycles and NADPH consumption [21]. NADPH consumption at the end of 15 and/or 30 min with 1–6 mM MSC concentration.

Figure 2. Kynurenine aminotransferase 1 (KYAT1) and thioredoxin reductase 1 (TrxR1) coupled activity assay to detect methylselenol generation. (a) TrxR1 activity in the coupled activity assay mixture. The assay mixture contains 5 mM Se-methylselenocysteine (MSC) as a primary substrate and 100 ng of KYAT1 as a primary enzyme to metabolize MSC into methylselenol. Methylselenol was utilized as a substrate by TrxR1, which is monitored by a decrease in absorbance at 340 nm with time in a spectrophotometer (oxidation of NADPH to NADP+). (b) The TrxR1 activity is represented in nmol of NADPH consumed/min. i.e., for 15, 30, 45 and 60 min. Data are represented as mean SD (n = 5). ± (c) TrxR1 activity with increased substrate concentration (MSC) with 100 ng of KYAT1. i.e., 1, 2, 4, 6, 8 and 10 mM of MSC, the Tmax velocity is >120, 83.6, 53.6, 35.0, 27.0 and 24.6 min respectively. (d) The enzyme activity with varying concentrations of the substrate at diﬀerent time points. With 100 ng of KYAT1 enzyme, the maximum activity can be observed at 15 min in all substrate concentrations. (e) TrxR1 activity at 30 min with increasing substrate concentration (MSC) in the presence of 200 ng of KYAT1 and 0.41µg of TrxR1. The apparent Km and Vmax for the TrxR1 coupled reaction was calculated to 5.84 0.95 mM and 1.12 0.08 nmol/min. Data are represented as mean SD (n = 4). ± ± ± Antioxidants 2020, 9, 139 6 of 14

Figure2b shows TrxR1 activity over time in this coupled activity assay. The following reaction is nonstoichiometric if there is access to oxygen and NADPH, i.e., most of the NADPH was consumed within 30 min. The consumption of NADPH increased with increased substrate concentration. The maximum time taken to consume all the NADPH was >120, 83.6, 53.6, 35.0, 27.0 and 24.6 min for 1,2,4,6,8 and 10 mM of MSC, respectively (Figure2c), and Figure2d shows the quick clearance of NADPH, i.e., there is only a little NADPH available after 45 min, so it is necessary to calculate the NADPH consumption at the end of 15 and/or 30 min with 1–6 mM MSC concentration.

3.2. Determination of Reaction Rate with Two Diﬀerent Substrates The coupled β-elimination assay/TrxR1 is kinetically complicated since it involves the activities of two enzymeswiththreedifferentsubstratesi.e., MSC,NADPH,andmonomethylselenol. In order tocharacterize the kinetics, the apparent Km and Vmax were calculated under saturated conditions. The apparent Km and Vmax were determined to 5.84 0.95 mM and 1.12 0.08 nmol/min respectively (Figure2e). Under these 1 ± ± conditions, a Kcat of 160 min− was calculated. We used the Kcat/Km value to calculate the turnover number 1 1 or catalytic efficiency of the enzyme. Under these conditions, the turnover number was 27.4 mM− min− .

3.3. Comparing KYAT1 Enzyme Activity in Transamination, β-Elimination/Pyruvate, and Coupled Activity Assay/TrxR1 To validate our coupled activity assay, we compared the enzyme activity with varying enzyme concentrations in transamination and coupled β-elimination assay/TrxR1. Figure3a,c show the activity of the enzyme with increasing enzyme concentration. Figure3a is a proof of our concept that TrxR1 uses methylselenol as a substrate and consume NADPH, as the enzyme concentration increases, the NADPH consumption also increases which reﬂects in the Tmax velocity. Comparable results were obtained from the transamination assay (Figure3c). As the coupled assay relies on NADPH availability, it is important to calculate the TrxR1 activity and/or MS generation at 15 or 30 min (Figure3b), but in case of transamination activity we determine the activity at 30 or 60 min (Figure3d), i.e., this assay measures the formation of PPA-enol and coupled β-elimination assay/TrxR1 measures the consumption of NADPH. From Figure3a,d, it is evident that the minimum amount of enzyme required for coupled activity assay/TrxR1 and transamination assay is 100 and 50 ng, respectively. Antioxidants 2020,, 9,, 139 7 of 1415

a b 340 -Blank A -Blank 340 TrxR1 Activity TrxR1 (nmol of NADPH of (nmol Sample A Sample consumed/min/mg of protein) of consumed/min/mg

c d 320 -Blank A -Blank 320 (µmol of PPA-enol of (µmol Sample Sample A formed/min/mg offormed/min/mg protein) KYAT1 KYAT1 Transamination Activity

Figure 3. Comparison between beta-elimination and transamination activity. (a) Increased KYAT1 concentration increases NADPH consumption by TrxR1. The Tmax velocity for 50, 100, 200 and 400 ng ofFigure KYAT1 3. Comparison enzymes resulted between in 49.71, beta‐elimination 49.68, 24.0 andand 14.65transamination min, respectively, activity. with (a) Increased 5 mM of MSCKYAT1 as aconcentration substrate. (b )increases The activity NADPH of the consumption KYAT1 enzyme by TrxR1. at varying The concentrations Tmax velocity for with 50, 100, time 200 in the and coupled 400 ng of KYAT1 enzymes resulted in 49.71, 49.68, 24.0 and 14.65 min, respectively, with 5mM of MSC1 as a activity assay. (c) The transamination activity of KYAT1 at varying enzyme concentration, 2 Tmax velocitysubstrate. for (b 50,) The 100, activity 200 and of the 400 KYAT1 ng of KYAT1enzyme enzymes at varying resulted concentrations in 78.15, with 53.0, time 46.4 in and the 31.7 coupled min, respectively.activity assay. Data (c) The are representedtransamination as mean activitySD of (KYAT1n = 3). (atd )varying The activity enzyme of the concentration, KYAT1 enzyme ½Tmax at ± varyingvelocity concentrationsfor 50, 100, 200 with and time 400 inng the of transaminationKYAT1 enzymes assay. resulted in 78.15, 53.0, 46.4 and 31.7 min, respectively. Data are represented as mean ± SD (n = 3). (d) The activity of the KYAT1 enzyme at 3.4. Comparingvarying concentrationsβ-Elimination with Activity time in Assay the transamination/Pyruvate with assay. a Coupled Activity Assay/TrxR1 The modiﬁed KYAT 1 β-elimination assay by J.L. Cooper et al. [15] was compared with our novel 3.4. Comparing β‐Elimination Activity Assay/Pyruvate with a Coupled Activity Assay/TrxR1 coupled activity assay for data reliability. From Figure4a,c,e, we observed that the activity of the enzyme was linearThe modified up to 140 KYAT ng for 1 β‐ theelimination coupled activity assay by assay J.L. /CooperTrxR1, 95et al. ng [15] for βwas-elimination compared assay with /ourpyruvate novel andcoupled 275 ngactivity for transamination assay for data assay.reliability. We assumeFrom Figure that the 4a,c,e, maximum we observed product that can the be activity calculated of the at thisenzyme concentration was linear of KYAT1—i.e.,up to 140 ng 0.13 for the0.005 coupled nmol of activity NADPH assay/TrxR1, consumed/min, 95 ng0.011 for β‐0.001elimination nmol of ± ± pyruvateassay/pyruvate formed and/min 275 and ng 1.08 for transamination0.02 nmol of PPA-enol assay. We formed assume/min that for the coupled maximum activity product assay /canTrxR1, be ± βcalculated-elimination at this assay concentration/pyruvate and of transamination KYAT1—i.e., 0.13 assay, ± 0.005 respectively. nmol of ToNADPH determine consumed/min, the feasibility 0.011 of our ± coupled0.001 nmol activity of pyruvate assay in formed/min crude-cell lysate, and 1.08 we transfected± 0.02 nmol a of KYAT1 PPA‐enol over-expressing formed/min plasmid for coupled into activity HEPG2 cellsassay/TrxR1, and harvested β‐elimination these cells assay/pyruvate after 48 h. We and used transamination 20 µg of protein assay, from respectively. the whole-cell To determine lysate from the bothfeasibility control of andour coupled KYAT1 over-expressed activity assay in cells. crude We‐cell observed lysate, we a clear transfected change a in KYAT1 NADPH over consumption‐expressing betweenplasmid into these HEPG2 two samples cells and (Figure harvested5a,b). TrxR1these cells activity after for 48 the h. controlWe used and 20 μ KYAT1g of protein over-expressed from the cellswhole was‐cell calculated lysate from to beboth 0.90 control and 2.31 andµ mol,KYAT1 respectively, over‐expressed of NADPH cells. consumed We observed/min a/mg clear of change protein in at theNADPH end of consumption 30 min. This between observation these indicates two samples that this (Figure coupled 5a,b). assay TrxR1 was activity a reliable for method the control for both and wholeKYAT1 cell over lysate‐expressed and pure cells KYAT was protein. calculated The to KYAT1 be 0.90 overexpression and 2.31 μmol, was respectively, veriﬁed by westernof NADPH blot (dataconsumed/min/mg not shown) of protein at the end of 30 min. This observation indicates that this coupled assay was a reliable method for both whole cell lysate and pure KYAT protein. The KYAT1 overexpression was verified by western blot (data not shown)

AntioxidantsAntioxidants2020, 9 2020, 139, 9, 139 9 of 158 of 14

Figure 4. Transamination and β-elimination activity of KYAT1 with inducers and inhibitors (a) Coupled activity assay at 15 min, TrxR1 activity with varying KYAT1 enzyme concentration. The optimum KYAT1Figure concentration 4. Transamination for maximum and β‐ NADPHelimination consumption activity of KYAT1 at 15 minwith isinducers 140 ng and (n = inhibitors7). (b) KYAT1 (a) Coupled activity assay at 15 min, TrxR1 activity with varying KYAT1 enzyme concentration. The activity (100ng) in coupled activity assay with phenylpyruvic acid (PPA) (400 µM), aminooxy acetic optimum KYAT1 concentration for maximum NADPH consumption at 15 min is 140 ng (n = 7). (b) acid (AOAA) (1 mM), l-tryptophan (l-Try) (2 mM) and auranofin (Aur) (100 nM) (n = 5–9). (c) The KYAT1 activity (100ng) in coupled activity assay with phenylpyruvic acid (PPA) (400 μM), aminooxy pyruvate production with varying KYAT1 enzyme concentrations by modified β-elimination assay at acetic acid (AOAA) (1 mM), L‐tryptophan (L‐Try) (2mM) and auranofin (Aur) (100 nM) (n = 5–9). (c) 60 min.The The pyruvate maximum production activity with was shownvarying at KYAT1 95 ng ofenzyme an enzyme concentrations concentration by modified (1/2Tmax β‐eliminationis 0.0125 nmol β of pyruvateassay at produced 60 min. The by 95ngmaximum of enzyme) activity (wasn = shown4). (d) at KYAT1 95 ng activityof an enzyme (200ng) concentration in -elimination (1/2Tmax assayis with phenylpyruvic0.0125 nmol of acid pyruvate (PPA) produced (400µM), by aminooxy 95ng of enzyme) acetic acid (n = (AOAA) 4). (d) KYAT1 (1 mM), activity indole-3-pyruvic (200ng) in β‐ acid (IPA) (100eliminationµM), l -tryptophanassay with phenylpyruvic (l-Try) (2 mM) acid and (PPA) BFF-122 (400μ (50M), µaminooxyM) (n = 4). acetic (e) Transaminationacid (AOAA) (1mM), activity of KYAT1indole enzyme‐3‐pyruvic at acid different (IPA) concentrations.(100 μM), L‐tryptophan The optimum (L‐Try) (2mM) KYAT1 and concentrationBFF‐122 (50 μM) for (n maximum= 4). (e) phenylpyruvate-enolTransamination formationactivity of atKYAT1 30 min enzyme was 274 at ng different (n = 4–5). concentrations. (f) Transamination The optimum activity KYAT1 of KYAT1 (50 ng) with phenylpyruvic acid (PPA) (400 µM), aminooxy acetic acid (AOAA) (1 mM), indole-3-pyruvic acid (IPA) (100 µM) and l-tryptophan (l-Try) (2 mM) (n = 4). Bar graphs in (b)(d) and (f) represent mean SD, statistical analysis performed with one-way ANOVA with 95% confidential interval ± followed by Dunnett multiple comparison test (ns= not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with control). Antioxidants 2020, 9, 139 10 of 15

concentration for maximum phenylpyruvate‐enol formation at 30 min was 274 ng (n = 4–5). (f) Transamination activity of KYAT1 (50 ng) with phenylpyruvic acid (PPA) (400 μM), aminooxy acetic acid (AOAA) (1 mM), indole‐3‐pyruvic acid (IPA) (100 μM) and L‐tryptophan (L‐Try) (2mM) (n = 4). Bar graphs in (b) (d) and (f) represent mean ± SD, statistical analysis performed with one‐way ANOVA with 95% confidential interval followed by Dunnett multiple comparison test (ns= not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with control). Antioxidants 2020, 9, 139 9 of 14

Figure 5. TrxR1 activity with KYAT1 over-expressed and mock transfected HEPG2 cells. (a) TrxR1 activity in cell lysates from control and KYAT1 over-expressed HEPG2 cells (20 µg of control and KYAT1 Figureover-expressed 5. TrxR1 cellactivity lysate). with The KYAT1 1/2Tmax overvelocity‐expressed for control and mock and KYAT1 transfected overexpressed HEPG2 cells. cell lysate(a) TrxR1 was activity>120 and in 48.04 cell lysates min. (b from) The control TrxR1 activity and KYAT1 at the endover of‐expressed 30 min for HEPG2 control cells and KYAT1(20 μg over-expressedof control and KYAT1cell lysate over is‐ 0.20expressed and 0.52 cell nmol lysate)./min The or 0.901/2Tmax0.16 velocity and 2.31for control0.10 µandmol KYAT1/min/mg overexpressed of protein. Graph cell ± ± lysaterepresent was mean >120 andSD, 48.04 statistical min. ( analysisb) The TrxR1 performed activity with at student the end t-test of 30 with min 95% for control confidential and KYAT1 interval ± over(*** p‐expressed< 0.001 compared cell lysate with is 0.20 control). and 0.52 nmol/min or 0.90 ± 0.16 and 2.31 ± 0.10 μmol/min/mg of protein. Graph represent mean ± SD, statistical analysis performed with student t‐test with 95% 3.5. Coupledconfidential Activity interval/TrxR1 (*** Assayp < 0.001 with compared Different with Inhibitors control). and Inducers Since the coupled activity assay involves two enzyme activities, we aimed to elucidate the 4.involvement Discussion of both enzyme activities by using inhibitors for the individual enzyme. Auranofin (Aur) at 100nMSe‐methylselenocysteine is known to inhibit TrxR1(MSC) activity is one of completely the most studied [22]. Aminooxyacetic selenium compounds acid (AOAA) because at 1of mM its highhas been bioavailability, reported to inhibitexcellent KYAT1 pharmacokinetic activity [23,24]. Phenylpyruvicproperties and acid, pronounced a 2-oxo acid, tumor was shown‐specific to cytotoxicityincrease the due activity to its of metabolites. the KYAT1 enzyme MSC as [9 ,a15 prodrug]. Additions requires of 100 enzymatic nM auranofin activity completely to transform abrogated into anthe active enzymatic drug. reaction KYAT1 while plays AOAA a crucial showed role in a metabolizing 50% decline in MSC enzyme into activity.an active However, drug. KYAT1 the addition is highly of expressed400 µM of phenylin a metabolically pyruvic acid active increased organs the such reaction as the rate liver, threefold. kidney Inhibitory and brain eff ectsand oftrace Aur amounts and AOAA of expressionwas reproducible are also with seen the in continuous the spleen, coupled heart, activitylarge intestine, assay. Inhibitors thyroid, and lung inducers and small showing intestine 200–300% [23]. KYAT1activity is with mostly PPA, found 40–50% in the activity cytosol with and AOAA, possesses and strong 0–10% aminotransferase activity with Aur activity (Figure towards4b) compared a wide varietywith control of amino (100 acid ng of substrates pure protein). such These as glutamine, inhibitors phenylalanine, responded similarly leucine, to kynurenine, the β-elimination tryptophan, activity methionine,assay except tyrosine, BFF-122 (Figurecysteine,4d). asparagine Meanwhile, and KYAT1 histidine inhibitors [15]. Several such as publications PPA, AOAA, have IPA, shown and l- TRYthe antishowed‐tumor 40–50%, and chemo 50–75%,‐preventive 10–20% effects and 10–15% of MSC reduced metabolites enzyme produced activity by in KYAT1, transamination metabolites activity such asassay methylselenol respectively(Figure and β‐methylselenopyruvate4f). The interesting candidate (MSP) from[2,25–27]. Figure Although4b,d,f is PPA, there which are several increases assay the methodsβ-elimination available activity to todetermine threefold the and transamination inhibits the transamination product of activityKYAT1, twofold. there exist only a few available technically complicated indirect assays with questionable accuracy to determine the β‐ 4. Discussion elimination product, i.e., methylselenol. TheSe-methylselenocysteine primary goal of the present (MSC) study is one was of the to mostestablish studied a simple selenium and compoundsreliable protocol because to detect of its andhigh measure bioavailability, methylselenol excellent pharmacokineticgeneration with propertiesa simple andspectrophotometric pronounced tumor-specific method. Until cytotoxicity now, methylselenoldue to its metabolites. has been MSC measured as a prodrug via laser requires desorption enzymatic ionization activity‐mass to transformspectrometry into (LDI an active‐MS), drug.GC‐ MSKYAT1 or other plays high a crucial throughput role in metabolizing techniques [8,21]. MSC intoThe anDNPH active method drug. KYAT1was used is highly to measure expressed the β‐ in eliminationa metabolically activity active spectrophotometrically. organs such as the liver, This kidney assay is and an brainindirect and measure trace amounts of pyruvate of expression and not are also seen in the spleen, heart, large intestine, thyroid, lung and small intestine [23]. KYAT1 is mostly found in the cytosol and possesses strong aminotransferase activity towards a wide variety of amino acid substrates such as glutamine, phenylalanine, leucine, kynurenine, tryptophan, methionine, tyrosine, cysteine, asparagine and histidine [15]. Several publications have shown the anti-tumor and chemo-preventive effects of MSC metabolites produced by KYAT1, metabolites such as methylselenol and β-methylselenopyruvate (MSP) [2,25–27]. Although there are several assay methods available to Antioxidants 2020, 9, 139 10 of 14 determine the transamination product of KYAT1, there exist only a few available technically complicated indirect assays with questionable accuracy to determine the β-elimination product, i.e., methylselenol. The primary goal of the present study was to establish a simple and reliable protocol to detect and measure methylselenol generation with a simple spectrophotometric method. Until now, methylselenol has been measured via laser desorption ionization-mass spectrometry (LDI-MS), GC-MS or other high Antioxidantsthroughput 2020 techniques, 9, 139 [8,21]. The DNPH method was used to measure the β-elimination activity11 of 15 spectrophotometrically. This assay is an indirect measure of pyruvate and not the production of the production of methylselenol [6]. So, in this current study, we modified the established β‐ methylselenol [6]. So, in this current study, we modified the established β-elimination assay and coupled elimination assay and coupled it with TrxR1. By combining these two known enzyme reactions into it with TrxR1. By combining these two known enzyme reactions into one assay, we created an assay one assay, we created an assay detecting methylselenol generation spectrophotometrically. KYAT1 detecting methylselenol generation spectrophotometrically. KYAT1 utilizes MSC as a substrate and utilizes MSC as a substrate and produces methylselenol via β‐elimination activity, and methylselenol produces methylselenol via β-elimination activity, and methylselenol reacts rapidly with oxygen to form reacts rapidly with oxygen to form methylseleninate (CH3Se− + O2  CH3SeO2−) [13]. Thioredoxin methylseleninate (CH3Se− + O2 CH3SeO2−)[13]. Thioredoxin reductases utilize methylseleninate as reductases utilize methylseleninate→ as a substrate and reduce it to CH3Se‐ at the expense of 2NADPH. a substrate and reduce it to CH Se- at the expense of 2NADPH. The following reaction was proposed The following reaction was proposed3 by Gromer S and Gross J.H (Figure 6) [13]. by Gromer S and Gross J.H (Figure6)[13]. NADPH + H++ CH3SeO2− NADP+ + CH3SeO− +H2O (Eq.1) NADPH + H++ CH3SeO−  NADP+ + + CH3Se− + H2O (Eq.2)+ NADPH + H + CH3SeO2− NADP + CH3SeO− +H2O (1) The net reaction will be → + 3 2−  + + 3 − +2 2NADPH + 2H + NADPHCH SeO + H 2NADP+ CH3SeO + CH− SeNADP + 2H O+ (Eq.3)CH3Se − + H2O (2) →

Figure 6. Schematic representation for the proposed mechanism of TrxR activity in reducing Figuremethylseleninate 6. Schematic [13]. representation for the proposed mechanism of TrxR activity in reducing methylseleninate [13]. The net reaction will be Similar TrxR1 activity with selenocystine (SeC) has previously been shown [19,28,29]. We + + 2NADPH + 2H + CH SeO − 2NADP + CH Se− + 2H O (3) assume that a similar kind of reaction might3 happen2 → with the MSC metabolite,3 2 i.e., MS. During the reaction, NADPH is oxidized to NADP+, which is detected by a decrease in absorbanceSimilar at TrxR1 340 nm activity with with time selenocystine in a spectrophotometer. (SeC) has previously From the been above shown equations, [19,28 ,we29]. can We assume assume that a similar kind of reaction might happen with the MSC metabolite, i.e., MS. that 2 moles of NADPH result in 1 mole of CH3SeO2−. An increase in substrate concentration resulted + in rapidDuring NADPH the reaction, consumption, NADPH i.e., is oxidized higher MSC to NADP concentration, which is results detected in by increased a decrease MS in generation. absorbance Ourat 340 data nm clearly with time show in athat spectrophotometer. the reaction rate Fromis proportional the above equations,to the MSC we concentration. can assume that The 2 herein moles describedof NADPH coupled result inassay 1 mole is kinetically of CH3SeO complicated2−. An increase since init reflects substrate the concentration activities of two resulted enzymes in rapid and NADPH consumption, i.e., higher MSC concentration results in increased MS generation. Our data three substrates. Comparisons of the apparent Km and Vmax with previously published data clearly show that the reaction rate is proportional to the MSC concentration. The herein described concerning selenite reveal that the apparent Km is considerably higher (Km = 5.84 mM for MSC) coupled assay is kinetically complicated since it reflects the activities of two enzymes and three compared to selenite (Km = 20 μM). Compared to selenocystine, the Kcat is much lower (160 min−1 for MSCsubstrates. and 3200 Comparisons min−1 for SeC) of the[29,30]. apparent These K kineticm and differences Vmax with previouslyare expected published due to the data high concerning reactivity ofselenite selenite reveal and seleneocystine. that the apparent MSC Km aloneis considerably without the higher KYAT1 (K enzymem = 5.84 in mM the assay for MSC) mixture compared does not to µ 1 changeselenite in (K absorbancem = 20 M). compared Compared to to the selenocystine, blank (Figure the S1a). Kcat Thisis much shows lower that (160 the rate min −of forour MSC coupled and 1 activity3200 min assay− for SeC)with [MSC29,30 ].as These a substrate kinetic didependsfferences on are KYAT1 expected metabolism. due to the high There reactivity is no ofliterature selenite availableand seleneocystine. to show that MSC MSP alone (transamination without the product) KYAT1 enzyme is a substrate in the assayfor TrxR1. mixture does not change in absorbanceThe detection compared limit to of the KYAT1 blank (Figure in the assay S1a). Thiswas shows100 ng, that since the there rate ofis ournot coupledmuch difference activity assayseen betweenwith MSC 50 as and a substrate 100 ng of depends KYAT1. on NADPH KYAT1 consumption metabolism. was There in is the no detectable literature availablerange from to 100 show to that400 ngMSP of (transaminationKYAT1. NADPH product) consumption is a substrate in the coupled for TrxR1. assay reaction depends on the concentration of KYAT1The and detection substrate limit (MSC) of KYAT1 respectively. in the assayApart was from 100 the ng, simplicity since there of the is notassay, much another difference advantage seen ofbetween this assay 50 andis that 100 it ng requires of KYAT1. a minimum NADPH amount consumption of enzyme was compared in the detectable to the DNPH range method. from 100 But to with400 ng transamination of KYAT1. NADPH activity, consumption KYAT1 is inmore the coupledcompetent, assay even reaction at 50 dependsng, because on the it was concentration reported in of several publications that KYAT1 favors transamination over β‐elimination with a 4:1 ratio [6,15] when sulfur‐substrates are used, i.e., KYAT1 is more efficient in transamination of phenylalanine to phenylpyruvate‐enol. For the modified β‐elimination assay, we have changed the absorbance from 420 to 515, because at this wavelength we observe high linearity with pyruvic acid standard (data not shown) and reduced background noises with colored compounds [18]. So as to make sure that the change in absorbance is because of MS generation rather than protein oxidation itself, we changed the substrate from MSC to L‐Phe in coupled activity assay (L‐Phe is known to be an excellent substrate of KYAT1 for transamination) [15] and we did not observe any changes in absorbance (Figure S1b) Antioxidants 2020, 9, 139 11 of 14

KYAT1 and substrate (MSC) respectively. Apart from the simplicity of the assay, another advantage of this assay is that it requires a minimum amount of enzyme compared to the DNPH method. But with transamination activity, KYAT1 is more competent, even at 50 ng, because it was reported in several publications that KYAT1 favors transamination over β-elimination with a 4:1 ratio [6,15] when sulfur-substrates are used, i.e., KYAT1 is more eﬃcient in transamination of phenylalanine to phenylpyruvate-enol. For the modiﬁed β-elimination assay, we have changed the absorbance from 420 to 515, because at this wavelength we observe high linearity with pyruvic acid standard (data not shown) and reduced background noises with colored compounds [18]. So as to make sure that the change in absorbance is because of MS generation rather than protein oxidation itself, we changed the substrate from MSC to l-Phe in coupled activity assay (l-Phe is known to be an excellent substrate of KYAT1 for transamination) [15] and we did not observe any changes in absorbance (Figure S1b) compared to blank. This is clear evidence that the decrease in absorbance is because of MSC metabolism into MS. According to eq.3, the amount of MS can be calculated from Figure4a, i.e., 140 ng of enzyme consumes 0.13 0.005 nmol of NADPH/min, which can be written as 0.065 0.0027 nmol of MS ± ± generated/min. A complicating factor is that MS can redox cycle with oxygen and a TrxR1 and thus the assay is not proportional to the amount of MSC/MS. However, our data clearly show that the consumption of NADPH was proportional to the amount of enzyme with the detection limit of 100 ng. To further characterize and validate the method, we used chemical modifiers for TrxR1 and KYAT1 activities respectively. To show the accuracy of the assay we used both KYAT1 and TrxR1 inhibitors. Auranofin (Aur) is known to be an effective inhibitor of TrxR1 [31], and 100 nM of Aur completely inhibits the TrxR1 activity [19]. In our study, we observed a complete cessation of NADPH consumption with the addition of 100 nM Aur. On the contrary, the KYAT1 inhibitor AOAA showed a 50% reduced NADPH consumption. AOAA has been shown to inhibit pyridoxal phosphate-dependent enzymes, such as KYAT1, effectively at 1 mM [23,24]. AOAA inhibited the enzyme activity in both the β-elimination and transamination assay. Inhibition was not limited to β-elimination activity, as previously reported by Elfarra et al. (1986), and Stevens et al. (1989) [24,32]. Phenyl pyruvic acid (PPA) was shown to increase the activity of KYAT1 [9,15]. We have shown in this study that PPA increased the enzyme activity towards MSC about 3–4-fold in the coupled activity assay/TrxR1 and 1.5-fold in the β-elimination assay/pyruvate (Figure4d). Interestingly, it specifically increased the KYAT1 β-elimination activity, whereas it partially inhibited (40–50%) the transamination activity. Other inhibitors, such as BFF-122 [33], showed 1.5-fold increased activity rather than inhibiting the enzyme, in the β-elimination assay, but not in the coupled or transamination assay. α-ketoglutarate [34] did not show any inhibitory or inducing activity towards the KYAT1 enzyme reaction in either the transamination nor β-elimination assay (data not shown); α-ketoglutarate is known to increase the KYAT1 activity [17]. Indole-3-pyruvic acid [34,35] showed a 50% reduced activity in the β-elimination activity assay and 20% reduced activity in transamination activity assay; we could not observe these effects in the coupled assay because the color of this compound interferes with the assay. As l-tryptophan is known to be a competitive inhibitor for the KYAT1 enzyme [35], we observed a strong inhibitory effect in the β-elimination/pyruvate activity assay but mild inhibitory effects in the coupled and transamination activity assay, but the data were not significant. From this data, we hypothesize that the assay described herein can be plausible in the whole-cell lysate. As expected, we found a clear deviation in NADPH consumption between control and KYAT1 over-expressed HEPG2 whole-cell lysate. TrxR1 activity for control and KYAT1 over-expressed cell lysate was, respectively, 0.9 0.16 and 2.31 0.10 µmol of NADPH consumed/min/mg of protein. ± ± From these data, we assume that the β-elimination activity in the control and KYAT1 over-expressed cells could be, respectively, 0.45 and 1.155 µmol/min/mg of protein at 30 min. We have observed threefold increases in pyruvate production at 120 min in KYAT1 over-expressed cell lysate compared to control in the β-elimination assay (Figure S2b), and a similar increase in transamination activity was also observed in these cell extracts (Figure S2a). Antioxidants 2020, 9, 139 12 of 14

In summary, the data presented herein show a simple and accurate method to determine KYAT1 β-elimination activity via spectrophotometer for both pure KYAT1 protein and whole-cell lysate. This assay is highly relevant for research of MSC, a selenium compound with outstanding potential for cancer prevention and treatment.

5. Conclusions In conclusion, our data presented here show a new way of quantifying and detecting β-elimination activity by pure KYAT1 protein and whole cell lysate by combining KYAT1 and TrxR1. TrxR1 redox cycle with methylselenol and oxidizing NADPH to NADP+, which was observed spectrophotometrically at 340 nm.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/2/139/s1, Figure S1: Coupled activity assay (a) With and without KYAT1 (100 ng) enzyme (b) with l-Phe and MSC as a substrate with 100 ng of KYAT1 enzyme, Figure S2: KYAT1 enzyme activity in Mock and KYAT1 overexpressed HEPG2 cells (a) Transamination activity at the end of 30 min in 20 µg of cell lysate (b) β-elimination activity at the end of 120min in 20 µg of cell lysate. Author Contributions: M.B. and A.K.S. conceived the study design; A.K.S. performed the experiments, data analyses, and presentation; M.B. and A.K.S. wrote the manuscript. A.K.S. was responsible for the ﬁnal editing of the entire manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Cancerfonden (180429), Cancer och Allergifonden (206), Radiumhemmets Forskningsfonder (171023) and Karolinska Institutet. Conﬂicts of Interest: Mikael Björnstedt is listed as the inventor of a patent application concerning the use of intravenous inorganic selenium in the treatment of cancer patients. Mikael Björnstedt is also a shareholder in a company, SELEQ OY, which develops selenium formulations for supplements and pharmaceutical formulations for the treatment of cancer.

References

1. Gabel-Jensen, C.; Lunoe, K.; Gammelgaard, B. Formation of methylselenol, dimethylselenide and dimethyldiselenide in in vitro metabolism models determined by headspace GC-MS. Met. Integr. Biometal Sci. 2010, 2, 167–173. [CrossRef][PubMed] 2. Pinto, J.T.; Lee, J.I.; Sinha, R.; MacEwan, M.E.; Cooper, A.J. Chemopreventive mechanisms of alpha-keto acid metabolites of naturally occurring organoselenium compounds. Amino Acids 2011, 41, 29–41. [CrossRef] [PubMed] 3. Commandeur, J.N.; Andreadou, I.; Rooseboom, M.; Out, M.; de Leur, L.J.; Groot, E.; Vermeulen, N.P. Bioactivation of selenocysteine Se-conjugates by a highly puriﬁed rat renal cysteine conjugate beta-lyase/glutamine transaminase K. J. Pharmacol. Exp. Ther. 2000, 294, 753–761. [PubMed] 4. Rooseboom, M.; Vermeulen, N.P.; Andreadou, I.; Commandeur, J.N. Evaluation of the kinetics of beta-elimination reactions of selenocysteine Se-conjugates in human renal cytosol: Possible implications for the use as kidney selective prodrugs. J. Pharmacol. Exp. Ther. 2000, 294, 762–769. 5. Rooseboom, M.; Vermeulen, N.P.E.; van Hemert, N.; Commandeur, J.N.M. Bioactivation of Chemopreventive Selenocysteine Se-Conjugates and Related Amino Acids by Amino Acid Oxidases Novel Route of Metabolism of Selenoamino Acids. Chem. Res. Toxicol. 2001, 14, 996–1005. [CrossRef] 6. Stevens, J.L.; Robbins, J.D.; Byrd, R.A. A puriﬁed cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K. J. Biol. Chem. 1986, 261, 15529–15537. 7. Lee, J.I.; Nian, H.; Cooper, A.J.; Sinha, R.; Dai, J.; Bisson, W.H.; Dashwood, R.H.; Pinto, J.T. Alpha-keto acid metabolites of naturally occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells. Cancer Prev. Res. 2009, 2, 683–693. [CrossRef] 8. Andreadou, I.; Menge, W.M.; Commandeur, J.N.; Worthington, E.A.; Vermeulen, N.P. Synthesis of novel Se-substituted selenocysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: Evaluation of kinetics of beta-elimination reactions in rat renal cytosol. J. Med. Chem. 1996, 39, 2040–2046. [CrossRef] Antioxidants 2020, 9, 139 13 of 14

9. Cooper, A.J.; Pinto, J.T. Aminotransferase, L-amino acid oxidase and beta-lyase reactions involving L-cysteine S-conjugates found in allium extracts. Relevance to biological activity? Biochem. Pharmacol. 2005, 69, 209–220. [CrossRef] 10. Zeng, H.W. Selenium as an Essential Micronutrient: Roles in Cell Cycle and Apoptosis. Molecules 2009, 14, 1263–1278. [CrossRef] 11. Ip, C.; Zhu, Z.; Thompson, H.J.; Lisk, D.; Ganther, H.E. Chemoprevention of mammary cancer with Se-allylselenocysteine and other selenoamino acids in the rat. Anticancer Res. 1999, 19, 2875–2880. [PubMed] 12. Ip, C.; Hayes, C.; Budnick, R.M.; Ganther, H.E. Chemical form of selenium, critical metabolites, and cancer prevention. Cancer Res. 1991, 51, 595–600. [PubMed] 13. Gromer, S.; Gross, J.H. Methylseleninate is a substrate rather than an inhibitor of mammalian thioredoxin reductase. Implications for the antitumor effects of selenium. J. Biol. Chem. 2002, 277, 9701–9706. [CrossRef] [PubMed] 14. Holmgren, A. Antioxidant function of thioredoxin and glutaredoxin systems. Antioxid. Redox Signal. 2000, 2, 811–820. [CrossRef] 15. Cooper, A.J.L.; Pinto, J.T.; Krasnikov, B.F.; Niatsetskaya, Z.V.; Han, Q.; Li, J.Y.; Vauzour, D.; Spencer, J.P.E. Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase. Arch. Biochem. Biophys. 2008, 474, 72–81. [CrossRef] 16. Cooper, A.J.L. Purification of soluble and mitochondrial glutamine transaminase K from rat kidney: Use of a sensitive assay involving transamination between l-phenylalanine and α-keto-γ-methiolbutyrate. Anal. Biochem. 1978, 89, 451–460. [CrossRef] 17. Cooper, A.J.; Krasnikov, B.F.; Pinto, J.T.; Bruschi, S.A. Measurement of cysteine S-conjugate beta-lyase activity. Curr. Protoc. Toxicol. 2010, 44.[CrossRef] 18. Anthon, G.E.; Barrett, D.M. Modified method for the determination of pyruvic acid with dinitrophenylhydrazine in the assessment of onion pungency. J. Sci. Food Agric. 2003, 83, 1210–1213. [CrossRef] 19. Cunniff, B.; Snider, G.W.; Fredette, N.; Hondal, R.J.; Heintz, N.H. A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates. Anal. Biochem. 2013, 443, 34–40. [CrossRef] 20. Arner, E.S.; Holmgren, A. Measurement of thioredoxin and thioredoxin reductase. Curr. Protoc. Toxicol. 2001, 24.[CrossRef] 21. Fernandes, A.P.; Wallenberg, M.; Gandin, V.; Misra, S.; Tisato, F.; Marzano, C.; Rigobello, M.P.; Kumar, S.; Bjornstedt, M. Methylselenol formed by spontaneous methylation of selenide is a superior selenium substrate to the thioredoxin and glutaredoxin systems. PLoS ONE 2012, 7, e50727. [CrossRef] 22. Marzano, C.; Gandin, V.; Folda, A.; Scutari, G.; Bindoli, A.; Rigobello, M.P. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic. Biol. Med. 2007, 42, 872–881. [CrossRef][PubMed] 23. Rooseboom, M.; Vermeulen, N.P.; Groot, E.J.; Commandeur, J.N. Tissue distribution of cytosolic beta-elimination reactions of selenocysteine Se-conjugates in rat and human. Chem. Biol. Interact. 2002, 140, 243–264. [CrossRef] 24. Elfarra, A.A.; Jakobson, I.; Anders, M.W. Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity. Biochem. Pharmacol. 1986, 35, 283–288. [CrossRef] 25. Cooper, A.J.; Krasnikov, B.F.; Niatsetskaya, Z.V.; Pinto, J.T.; Callery, P.S.; Villar, M.T.; Artigues, A.; Bruschi, S.A. Cysteine S-conjugate beta-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents. Amino Acids 2011, 41, 7–27. [CrossRef] [PubMed] 26. Rooseboom, M.; Schaaf, G.; Commandeur, J.N.; Vermeulen, N.P.; Fink-Gremmels, J. Beta-lyase-dependent attenuation of cisplatin-mediated toxicity by selenocysteine Se-conjugates in renal tubular cell lines. J. Pharmacol. Exp. Ther. 2002, 301, 884–892. [CrossRef][PubMed] 27. Nian, H.; Bisson, W.H.; Dashwood, W.M.; Pinto, J.T.; Dashwood, R.H. Alpha-keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells. Carcinogenesis 2009, 30, 1416–1423. [CrossRef][PubMed] 28. Bjornstedt, M.; Hamberg, M.; Kumar, S.; Xue, J.; Holmgren, A. Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH and selenocystine strongly stimulates the reaction via catalytically generated selenols. J. Biol. Chem. 1995, 270, 11761–11764. [CrossRef] Antioxidants 2020, 9, 139 14 of 14

29. Bjornstedt, M.; Kumar, S.; Bjorkhem, L.; Spyrou, G.; Holmgren, A. Selenium and the thioredoxin and glutaredoxin systems. Biomed. Environ. Sci. BES 1997, 10, 271–279. 30. Kumar, S.; Bjornstedt, M.; Holmgren, A. Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen. Eur. J. Biochem. FEBS 1992, 207, 435–439. [CrossRef] 31. Gandin, V.; Fernandes, A.P.; Rigobello, M.P.; Dani, B.; Sorrentino, F.; Tisato, F.; Bjornstedt, M.; Bindoli, A.; Sturaro, A.; Rella, R.; et al. Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase. Biochem. Pharmacol. 2010, 79, 90–101. [CrossRef][PubMed] 32. Stevens, J.L.; Hatzinger, P.B.;Hayden, P.J.Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate beta-lyase. Drug Metab. Dispos. 1989, 17, 297–303. [PubMed] 33. Rossi, F.; Valentina, C.; Garavaglia, S.; Sathyasaikumar, K.V.; Schwarcz, R.; Kojima, S.; Okuwaki, K.; Ono, S.;

Kajii, Y.; Rizzi, M. Crystal structure-based selective targeting of the pyridoxal 50-phosphate dependent enzyme kynurenine aminotransferase II for cognitive enhancement. J. Med. Chem. 2010, 53, 5684–5689. [CrossRef] 34. Han, Q.; Li, J.; Li, J. pH dependence, substrate speciﬁcity and inhibition of human kynurenine aminotransferase I. Eur. J. Biochem. 2004, 271, 4804–4814. [CrossRef] 35. Han, Q.; Robinson, H.; Cai, T.; Tagle, D.A.; Li, J. Structural insight into the inhibition of human kynurenine aminotransferase I/glutamine transaminase K. J. Med. Chem. 2009, 52, 2786–2793. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).