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A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase

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A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase life Article A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase Angela Tramonti 1,2,† , Roberto Contestabile 2,† , Rita Florio 3, Caterina Nardella 2, Anna Barile 1,2 and Martino L. Di Salvo 2,* 1 Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Piazzale Aldo Moro 5, 00185 Roma, Italy; [email protected] (A.T.); [email protected] (A.B.) 2 Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, P.le A. Moro, 5, 00185 Roma, Italy; [email protected] (R.C.); [email protected] (C.N.) 3 European Brain Research Institute, Fondazione “Rita Levi-Montalcini”, 00185 Roma, Italy; r.fl[email protected] * Correspondence: [email protected] † Both authors equally contributed to this work. Abstract: Cysteine sulfinic acid decarboxylase catalyzes the last step of taurine biosynthesis in mammals, and belongs to the fold type I superfamily of pyridoxal-50-phosphate (PLP)-dependent enzymes. Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal tissues; it is highly present in liver, kidney, muscle, and brain, and plays numerous biological and physiological roles. Despite the importance of taurine in human health, human cysteine sulfinic acid decarboxylase has been poorly characterized at the biochemical level, although its three-dimensional structure has been solved. In the present work, we have recombinantly expressed and purified human cysteine sulfinic acid decarboxylase, and applied a simple spectroscopic direct method based on circular dichroism to measure its enzymatic activity. This method gives a significant advantage in terms of simplicity and reduction of execution time with respect to previously used assays, Citation: Tramonti, A.; Contestabile, and will facilitate future studies on the catalytic mechanism of the enzyme. We determined the R.; Florio, R.; Nardella, C.; Barile, A.; kinetic constants using L-cysteine sulfinic acid as substrate, and also showed that human cysteine Di Salvo, M.L. A Novel, Easy Assay Method for Human Cysteine Sulfinic sulfinic acid decarboxylase is capable to catalyze the decarboxylation—besides its natural substrates Acid Decarboxylase. Life 2021, 11, 438. L-cysteine sulfinic acid and L-cysteic acid—of L-aspartate and L-glutamate, although with much https://doi.org/10.3390/life11050438 lower efficiency. 0 Academic Editor: Attila Ambrus Keywords: cysteine sulfinic acid decarboxylase; pyridoxal 5 -phosphate; circular dichroism; enzy- matic assay Received: 19 April 2021 Accepted: 6 May 2021 Published: 14 May 2021 1. Introduction Publisher’s Note: MDPI stays neutral Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal with regard to jurisdictional claims in tissues and is highly present in liver, kidney, muscle, and brain, accounting for 25%, 50%, published maps and institutional affil- 53%, and 19% of their free amino acid pools, respectively [1]. Many works have shown iations. that taurine plays numerous biological and physiological roles [2]. In fact, not only does it conjugate bile acids [3], but it also plays a role as antioxidant [4,5], osmoregulator [6], and membrane stabilizer [7]. In the central nervous system, taurine acts as neurotransmitter, neuroprotective agent, and regulator of intracellular calcium homeostasis [8]. Low levels of Copyright: © 2021 by the authors. taurine are associated with various pathological conditions, including cardiomyopathy [9], Licensee MDPI, Basel, Switzerland. retinal degeneration [10], prenatal and postnatal growth retardation [11], and obesity [12]. This article is an open access article Thanks to its beneficial properties on human health, taurine is added in most infant distributed under the terms and milks [13], in energy drinks, and in cosmetics [14]. conditions of the Creative Commons Taurine derives from L-cysteine (Figure1). The major route for its synthesis is through Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ the decarboxylation of L-cysteine sulfinic acid (CSA) to hypotaurine by cysteine sulfinic 4.0/). acid decarboxylase (CSAD; EC 4.1.1.29), and the subsequent oxidation of hypotaurine to Life 2021, 11, 438. https://doi.org/10.3390/life11050438 https://www.mdpi.com/journal/life Life 2021, 11, x FOR PEER REVIEW 2 of 10 Life 2021, 11, 438 2 of 10 sulfinic acid decarboxylase (CSAD; EC 4.1.1.29), and the subsequent oxidation of hy- potaurine to taurine. An alternative pathway is the oxidation of CSA to L-cysteic acid (CA),taurine. followed An alternative by the decarboxylation pathway is the oxidation of CA to oftaurine CSA tocataly L-cysteiczed by acid a decarboxylase (CA), followed that by isthe to decarboxylation be CSAD as well of [15] CA. to Despite taurine the catalyzed importance by a decarboxylaseof taurine in human that is health, to be CSAD human as CSADwell [15 has]. Despite been poorly the importance characterized of taurine at the in biochemical human health, level. human It belongs CSAD to has the been aspartate poorly aminotransferasecharacterized at the fold biochemical type I super level.family It belongs of pyridoxal to the aspartate-5′-phosphate aminotransferase (PLP)-dependent fold enzymestype I superfamily [16]. Significantly, of pyridoxal-5 several0-phosphate other PLP (PLP)-dependent-dependent enzymes enzymes are [16involved]. Significantly, in tau- rineseveral biosynthesis other PLP-dependent (Figure 1). CSAD enzymes is expressed are involved in brain in and taurine liver from biosynthesis two tissue (Figure-specific1). mRNAsCSAD is that expressed differ inin braintheir and5′-untranslated liver from two regions tissue-specific due to an mRNAs alternative that differsplicing, in theiryet re- 50- sultuntranslated in two identical regions dueproteins to an [17] alternative. In vivo splicing, and in vitro yet result studies in twosuggested identical that proteins CSAD [ 17ac-]. tivityIn vivo increasesand in vitro whenstudies the suggestedenzyme thatis phosphorylated, CSAD activity increases and it whenis inhibited the enzyme when is dephosphorylated,phosphorylated, and with it isprotein inhibited kinase when C and dephosphorylated, protein phosphatase with 2C protein probably kinase involved C and inprotein this regulation phosphatase [18] 2C. Among probably PLP involved-dependent in this decarboxylases, regulation [18 ].CSAD Among shows PLP-dependent the strong- estdecarboxylases, homology with CSAD the shows two isoforms the strongest of glutamate homology decarboxylase, with the two isoforms GAD65 ofand glutamate GAD67 [17]decarboxylase,, responsible GAD65 for the and decarboxylation GAD67 [17], responsible of L-glutamate for the decarboxylation to produce γ-aminobutyrate of L-glutamate (GABA),to produce theγ -aminobutyratemain inhibitory (GABA),neurotransmitter the main of inhibitory the central neurotransmitter nervous system. of It the has central been shownnervous that system. GAD is It a hasble beento decarboxylate shown that both GAD CSA is able and to CA decarboxylate [15], whereas both no activity CSA and of CSADCA [15 was], whereas found nowith activity L-glutamate of CSAD [17] was. The found substrate with selectivity L-glutamate of [GAD17]. Theand substrateCSAD is determinedselectivity of by GAD the identity and CSAD of am isino determined acids occupying by the three identity specific of amino positions acids at occupying the active sitethree of specific the two positions enzymes at [19] the. active A taurine site of biosynthetic the two enzymes pathway [19 ].has A taurinealso been biosynthetic found in bacteria,pathway and has alsoCSAD been from found Synechococcus in bacteria, sp and. PCC CSAD 7335 from wasSynechococcus recombinantlysp. expressed PCC 7335 wasand purifiedrecombinantly [17]. On expressed the other and hand, purified taurine [17 seems]. On thenot otherto be hand,present taurine in plants, seems although not to be a novelpresent biosynthetic in plants, although pathway a rising novel from biosynthetic L-serine pathway was quite rising recently from identified L-serine wasin micro- quite algaerecently [20] identified. in microalgae [20]. Figure 1. Taurine biosynthetic pathway in mammals. In the present work, we have recombinantly expressed and purified purified human CSAD, and applied a simple spectroscopic, direct method to measure its enzyme activity with CSA and CA. This method gives a significant significant advantage in in terms of of simplicity and and re- re- duction o off execution time, with respect to previously used assays such as that based on o--phthalaldehydephthalaldehyde derivatizationderivatization of of products products and and high-performance high-performance liquid liquid chromatography chromatog- raphydetection detection [19]. We [19] also. We showed also thatshowed human that CSAD human is capable CSAD tois catalyzecapable theto cataly decarboxylationze the de- cofarboxylation L-aspartate of and L- L-glutamate,aspartate and although L-glutamate, with although much lower with efficiency. much lower efficiency. 2. Materials and Methods 2.1. Materials Ingredients for bacterial growth, chemicals, and enzyme substrates (CSA, CA, L- glutamate, and L-aspartate) in pure form were purchased from Sigma-Aldrich (St. Louis, Life 2021, 11, 438 3 of 10 MO, USA). HisTrap affinity columns (Ni-NTA) for purification of the 6xHis-tagged protein were purchased from GE Healthcare (Chicago, IL, USA). 2.2. Purification of CSAD The plasmid pNIC28-Bsa4 containing the cDNA
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