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Article A Novel, Easy Assay Method for Human Sulfinic 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 in mammals, and belongs to the fold type I superfamily of pyridoxal-50-phosphate (PLP)-dependent . Taurine (2-aminoethanesulfonic acid) is the most abundant free in animal tissues; it is highly present in , , 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 . We determined the R.; Florio, R.; Nardella, C.; Barile, A.; kinetic constants using L-cysteine sulfinic acid as , 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 —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 [3], but it also plays a role as [4,5], osmoregulator [6], and membrane stabilizer [7]. In the central nervous system, taurine acts as , neuroprotective agent, and regulator of intracellular [8]. Low levels of Copyright: © 2021 by the authors. taurine are associated with various pathological conditions, including cardiomyopathy [9], Licensee MDPI, Basel, Switzerland. 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 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 CSAD [ 17ac-]. tivityIn vivo increasesand in vitro whenstudies the suggested enzyme that is phosphorylated, CSAD activity increases and it when is inhibited the enzyme when is dephosphorylated,phosphorylated, and with it isprotein inhibited when C and dephosphorylated, 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 of and 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- 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 derivatization derivatization 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,

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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 of CSAD (UniProt entry code Q9Y600) was a gift from Nicola Burgess-Brown (Addgene plasmid # 42387). It is derived from the pET28a vector. In this vector, the cDNA of CSAD (GenBank: AAH98342.1) is fused to an N-terminal tag of 23 residues (MHHHHHHSSGVDLGTENLYFQS) including a hexahis- tidine (His6) and a TEV- cleavage site. The plasmid was transformed into E. coli Rosetta(DE3) cells, containing pLys helper plasmid. An overnight culture of these cells was used to inoculate 2 L of Vogel-Bonner medium E containing kanamycin (40 mg·L−1), (35 mg·L−1), and pyridoxine (30 mg·L−1). Bacteria were allowed to grow for approximately 5 h at 37 ◦C (until their ◦ OD600 reached ~0.6), then the growing temperature was lowered to 25 C and the expres- sion of CSAD was induced with 0.1 mM isopropyl thio-β-D-galactopyranoside (IPTG). Bacteria were harvested after 18 h of growth at 25 ◦C and suspended in 50 mL of 100 mM NaHEPES, pH 8, containing 500 mM NaCl, 5% glycerol, 0.2 mM dithiothreitol (DTT), and an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor (Sigma-Aldrich). Cell lysis was carried out by sonication on ice (5 min in short 10 s pulses, with 5 s intervals). Lysate was centrifuged at 12,000× g for 30 min to remove insoluble debris. The super- natant was loaded onto a column of Ni-NTA agarose resin pre-equilibrated with buffer A (20 mM NaHEPES, pH 7.5, containing 500 mM NaCl, 5% glycerol, and 0.2 mM DTT). The column was washed with 10 volumes of buffer A, 10 volumes of buffer A containing 10 mM , and eluted with buffer A containing 500 mM imidazole. Pooled fractions containing CSAD, as detected by SDS-PAGE analysis, were concentrated and washed in 20 mM NaHEPES, pH 7.5, containing 300 mM NaCl, 5% glycerol, and 0.2 mM DTT, using centrifuge ultrafiltration devices (Sartorius).

2.3. Spectroscopic Measurements All spectroscopic measurements were carried out at 20 ◦C in 20 mM NaHEPES buffer, pH 7.5, containing 300 mM NaCl, 0.2 mM DTT, and 5% glycerol. UV-visible spectra were recorded with a Hewlett-Packard 8453 diode-array spectrophotometer (Agilent Technolo- gies). The growth of bacterial cultures was monitored by determining the optical density at 600 nm (OD600), using the same diode array spectrophotometer. Protein subunit (molecular weight 57.576 kDa) concentration was calculated using an extinction coefficient at 280 nm of 63,576 M−1 cm−1 (calculated with the Gill and Von Hippel method; [21]). Circular dichroism measurements were carried out using a Jasco 710 spectropolarimeter. Acquisi- tion parameters were as follows: start 250 nm–end 190 nm; data pitch: 0.5 nm; scanning speed: 10 nm/min, response: 0.25 s; bandwidth: 2 nm; accumulation: 3.

2.4. Thin Layer Chromatography Samples of 2 µL of reaction mixtures were spotted on silica plates (Merck) with CSA, CA, glutamate, aspartate, and β- as standards. The mobile phase was a mixture of butanol:acetic acid:H2O in the 3:1:1 ratio. After chromatography, amino acids were revealed upon treatment with 0.5% in acetone, followed by heating at 90 ◦C.

2.5. Activity Assay of CSAD with CSA and CA The activity of CSAD versus CSA and CA was assayed by circular dichroism (CD) measurements. Decarboxylation of CSA and CA was followed by measuring the decrease of the CD signal at 220 nm due to the conversion of these L-amino acids into an achiral (taurine and hypotaurine, respectively). Far-UV (190–250 nm) CD spectra of CSA and other potential amino acid substrates (CA, L-glutamate, and L-aspartate), measured using a Jasco 710 spectropolarimeter equipped with a DP 520 processor and a 1 cm path length quartz cuvette, are shown in Figure 3b. All amino acids show a positive CD band, Life 2021, 11, 438 4 of 10

although of different intensity. CSAD has a negative CD band that does not interfere with the measurement since the enzyme concentration keeps constant during the assay. The reactions were started by adding 0.5 mM CSA or 2 mM CA to a solution of 0.65 µM CSAD in 20 mM potassium phosphate buffer, pH 7.2, containing 0.2 mM DTT, 0.1 mM EDTA, and 0.06 mM PLP. The decrease of CD signal at 220 nm was monitored over time. This wavelength was chosen so as to maximize the signal, while maintaining a linear relationship between CD signal and amino acid substrate concentration. The reaction rate was expressed as variation of ellipticity per minute (mdeg min−1).

2.6. Activity Assay of CSAD with Glutamate The reaction of CSAD with L-glutamate was assayed using GABase (Sigma-Aldrich, St. Louis, Mo, USA), a commercial preparation containing GABA aminotransferase and succinic semialdehyde dehydrogenase, which is normally employed to measure GAD activity [22]. CSAD (30 µM) was incubated at 30 ◦C with 50 mM sodium L-glutamate in 20 mM potassium phosphate buffer, pH 7.2, containing 0.2 mM DTT, 0.1 mM EDTA, and 0.6 mM PLP. Aliquots of this reaction mixture were taken at time intervals (5–90 min) and halted by boiling for 5 min. After centrifugation, each aliquot (50 µL) was mixed with 0.2 mL of GABase solution (0.1 M NaHEPPS, pH 8.6, containing 1 mM NADP+, 1 mM α-ketoglutarate, 3 mM mercaptoethanol, and 0.6 unit/mL of GABase), pre-activated at room temperature for 20 min. After 40 min at 37 ◦C, the reaction mixture was diluted to 1 mL with water, and the amount of NADPH formed—which is equimolar to the GABA produced—was determined by measuring the absorbance at 340 nm using an extinction coefficient of 6220 M−1cm−1.

2.7. Data Analysis All data analyses were carried out using the software Prism (GraphPad Software Inc., San Diego, CA, USA). Steady-state kinetic parameters were obtained by nonlinear least squares fitting of initial velocity data to the Michaelis–Menten equation.

3. Results 3.1. Expression, Purification, and Spectrophotometric Analysis of Human CSAD Human CSAD (EC 4.1.1.29), whose cDNA was cloned in pNIC28-Bsa4, was expressed in the E. coli Rosetta(DE3) strain with an N-terminal tag of 23 residues including a - stretch. The recombinant protein was purified to homogeneity, as judged by SDS-PAGE analysis (Figure2a, original figure is in Supplementary Materials). Although most of the expressed protein was found in the inclusion bodies (lane 4 vs. 3 in Figure2a), the purification yield was of about 3 mg of soluble protein per liter of bacterial culture. The absorption spectrum of the purified CSAD suggested that only a very small amount of PLP was bound to the protein. The addition of exogenous PLP—in equimolar concentration with respect to the protein—restored the holo-form of the enzyme, as demonstrated by the measurement of a neat increase in catalytic activity (see below for details on the assay method). After extensive dialysis, the presence in the absorption spectrum of two absorption bands at 330 and at 400 nm (Figure2b) suggested that PLP was retained by the protein, although absorption bands with maxima at these wavelengths usually correspond to free PLP [23]. However, aldimines absorbing between 385 and 400 nm have been reported in the literature in different PLP-dependent decarboxylases [24–26]. In order to keep the protein in the holo-form, excess PLP was added to the purified protein sample and to all reaction mixtures during activity assays. In these conditions, the protein was stable for several weeks at 4 ◦C. Life 2021, 11, x FOR PEER REVIEW 5 of 10

ature in different PLP-dependent decarboxylases [24–26]. In order to keep the protein in the holo-form, excess PLP was added to the purified protein sample and to all reaction Life 2021, 11, 438 mixtures during activity assays. In these conditions, the protein was stable for several5 of 10 weeks at 4 °C.

1 22 3 4 4 5 5 66 7

175 0.3 130 120 1.5 95 85 0.2 70 62 1.0 Absorbance 0.1 51 50 42 0.00 30030 350 400 450 500

Absorbance Wavelength (nm) 35 0.5 29

25 0.00 250 300 350 400 450 500 550 22 20 Wavelength (nm)

(a) (b)

FigureFigure 2. 2. PurificationPurification of of CSAD. CSAD. (a ()a )SDS-PAGE SDS-PAGE analysis analysis of of total total prot proteinein extract extract (2); (2); soluble soluble (3) (3) and and insolubleinsoluble (4) (4) fractions fractions of of lysate; lysate; 1 1μµgg (4) (4) and and 5 5μgµ g(5) (5) of of purified purified human human CSAD. CSAD. Molecular Molecular weight weight standardsstandards are are indicated in kDa (lane 1 andand 7).7). TheThe black lineline indicatesindicates thatthat thethe imageimage waswas assembledassem- bledfrom from two two different different gels, gels, whereas whereas the whitethe white line line separates separates different different parts parts of the of the same same gel gel that that were werecombined, combined, excluding excluding lanes lanes that that were were not ofnot interest; of interest; (b) Absorption(b) Absorption spectrum spectrum of purifiedof purified human humanCSAD. CSAD. UV–VIS UV–VIS absorption absorption spectrum spectrum was recorded was recorded in 20 mM in 20 NaHEPES mM NaHEPES buffer buffer pH 7.5, pH containing 7.5, containing300 mM NaCl, 300 mM 0.2 mM NaCl, DTT, 0.2 andmM 5% DTT, glycerol. and 5% glycerol.

3.2.3.2. Activity Activity Assay: Assay: Decarboxylation Decarboxylation of of CSA CSA and and CA CA TheThe activity ofof CSAD CSAD was was first first checked checked by thin-layerby thin-layer chromatography chromatography analysis. analysis. From FromFigure Figure3a, it is 3A, clear it thatis clear the purifiedthat the enzymepurified isenzyme able to is use able both to CSAuse andboth CA CSA as and substrates, CA as substrates,converting themconverting to hypotaurine them to andhypotaurin taurine,e respectively. and taurine, These respectively. results demonstrate These results that demonstraterecombinant that human recombinant CSAD is human active. CSAD In order is toactive. quantitatively In order to assayquantitatively the decarboxylase assay the decarboxylaseactivity of CSAD, activity we setof CSAD, up a method we set up based a method on CD based measurements. on CD measurements. This method This has methodbeen previously has been usedpreviously to determine used to the determin activitye ofthe other activity decarboxylases of other decarboxylases such as such asdecarboxylase [27] and diaminopimelate [27] and diaminopimelate decarboxylase decarboxylase [28]. It is based [28]. on It theis based fact that on theamino factacid that substratesamino acid have substrates a chiral have center a chiral (the α -carbon)center (the and α-carbon) show a far-UV and show CD spectruma far-UV CD(Figure spectrum3b), whereas (Figure their 3B), decarboxylationwhereas their decarb productsoxylation do not. products Therefore, do asnot. the Therefore, irreversible as thedecarboxylation irreversible decarboxylation reaction proceeds, reaction the CD proceed signal decreasess, the CD assignal a result decreases of the conversionas a result of thethe conversion amino acid of into the an amino achiral acid amine. into Asan shownachiral in amine. Figure As3c, shown the complete in Figure decarboxylation 3C, the com- pleteof 0.5 decarboxylation mM CSA (into hypotaurine), of 0.5 mM CSA monitored (into hypotaurine), at 220 nm in the monitored presence at of 0.66220 nmµM CSADin the ◦ presenceat 20 C, of was 0.66 accomplished μM CSAD at in20 less°C, was than accomplished 400 s. We also in less tested than 3 mM400 s. CA, We L-glutamate,also tested 3 −1 mMand L-aspartate,CA, L-glutamate, but we and observed L-aspartate, activity but only we withobserved CSA (activity−0.080 mdegonly with min CSAper (−µ0.080M of −1 µ mdegCSAD) min and−1 per CA μ (−M0.0045 of CSAD) mdeg and min CA per(−0.0045M of mdeg CSAD). min Possibly,−1 per μM with of CSAD). this method Possibly, and withunder this such method conditions, and theunder reaction such ratesconditio of L-glutamatens, the reaction and L-aspartate rates of L-glutamate decarboxylation and L-aspartatewere too low decarboxylation to be measured. were The too kinetics low to parametersbe measured. of CSAThe kinetics decarboxylation parameters were of CSAdetermined. decarboxylation In order towere convert determined. the observed In order ellipticity to convert changes the into observed the concentration ellipticity of CSA consumed in the decarboxylation reaction, a calibration curve was constructed, changes into the concentration of CSA consumed in the decarboxylation reaction, a cali- relating CSA concentration to CD signal. CD spectra of CSA at concentrations ranging bration curve was constructed, relating CSA concentration to CD signal. CD spectra of from 0.125 to 1 mM are shown in Figure4a. Figure4b shows that the CD signal at 220 nm CSA at concentrations ranging from 0.125 to 1 mM are shown in Figure 4A. Figure 4B was linear up to 1 mM CSA concentration. The initial rate of CSA decarboxylation was shows that the CD signal at 220 nm was linear up to 1 mM CSA concentration. The initial measured as a function of CSA concentration, obtaining a saturation curve from which a rate of CSA decarboxylation−1 was measured as a function of CSA concentration, obtaining kcat of 5.6 ± 0.2 s and a KM equal to 0.20 ± 0.02 mM were determined (Figure4c). a saturation curve from which a kcat of 5.6 ± 0.2 s−1 and a KM equal to 0.20 ± 0.02 mM were determined (Figure 4C).

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Figure 3. a FigureFigure 3. Decarboxylation3. DecarboxylationDecarboxylation of CSAof of CSA CSA and and andCA. CA. CA.(a) ( Thina) ( Thin) Thin-layer-layer-layer chromatography chromatography chromatography of reactionof ofreaction reaction Scheme Scheme Scheme 10. 10. 10. ◦ mM)mM)(mM) with with with CSAD CSAD CSAD (30 (30 μ (30M) μµM) atM) 30at at 30°C 30 °CforC for 1 for h 1 in 1h h50in in 50mM 50 mM mMpotassium potassium potassium phosphate phosphate phosphate buffer, buffer, buffer, pH pH pH7.5 7.5, 7.5,containing, containing containing 0.20.2 mM0.2 mM mM DTT, DTT, DTT, 0.1 0.1 0.1mM mM mM EDTA EDTA EDTA,, and, and 0.08 0.08 mM mM PLP. PLP. Samples SamplesSamples of ofCSA,of CSA,CSA, CA, CA,CA, hypotaurine hypotaurine,hypotaurine, and,and and taurine taurinetaurine were werewererun run asrun as references; asreferences; references; (b ()b CD )( bCD) spectra CD spectra spectra of of 0.5 0.5of mM 0.5 mM mM CSA CSA CSA (black (black (black line), line), line), CA CA (redCA (red line),(red line), line), L-glutamate L- glutamateL-glutamate (blue (blue (blue line), line)line)and, and, L-aspartateand L- aspartateL-aspartate (green (green (green line). line). line). AsAs As control,control, control, thethe the CD CD spectrum spectrum of of of3 3 µM µ3 MµM CSAD CSAD CSAD (dotted (dotted (dotted line) line) line) was was was also alsoalsorecorded; recorded recorded (;c ()c; The) (Thec) The decarboxylation decarboxylation decarboxylation reaction reaction reaction of of CSA CSAof CSA was was monitoredwas monitored monitored as as change aschange change of ellipticityof ellipticof ellipticity at 220ityat 220at nm 220 as nm as a function of time. The CD signal was measured as a function of time upon mixing CSAD nma function as a function of time. of Thetime. CD The signal CD signal was measured was measured as a function as a function of time of upon time mixingupon mixing CSAD CSAD (0.66 µ M) (0.66 μM) with 0.5 mM CSA in 20 mM potassium phosphate buffer, pH 7.2, containing 0.2 mM (0.66with μ 0.5M) mMwith CSA 0.5 mM in 20 CSA mM in potassium 20 mM pota phosphatessium phosphate buffer, pH buffer, 7.2, containing pH 7.2, containing 0.2 mM DTT,0.2 mM 0.1 mM DTT,DTT, 0.1 0.1 mM mM EDTA EDTA, and, and 0.06 0.06 mM mM PLP PLP at roomat room temperature. temperature. EDTA, and 0.06 mM PLP at room temperature.

FigureFigureFigure 4. Determination4 4.. DeterminationDetermination of kineticof of kinetic kinetic parameter parameter parameter for for forCSA CSA CSA decarboxylation decarboxylation decarboxylation by bycircular circular dichroism. dichroism. (a) ((a) a) CD CDCD spectraspectra spectra ofof CSACSAof CSA at at 0.125 0.125at 0.125 mM mM mM (green), (green), (green), 0.25 0.25 0.25 mM mM mM (red), (red), (red), 0.5 0.5 mM 0.5 mM (black),mM (black) (black) and, and, 1 and mM 1 mM 1 (blue);mM (blue); (blue); (b) (b) Dependence (b) De- De- pendencependenceof ellipticity of ellipticityof ellipticity (θ) at 220(θ )( θat nm) 220at as220 nm a nm function as asa function a function of CSA of CSAof concentration. CSA concentration. concentration. The Tθhe Twas heθ was θ measured was measured measured at differentat at differentdifferentconcentration concentration concentration of CSA. of CSA.of The CSA. fittingThe The fitting offitting data of dataof with data with a with linear a linear a regressionlinear regression regression gives gives agives value a value a value of 50of ±of50 0.7 50± 0 ±. mdeg7 0 .7 mdeg per 1 mM of CSA; (c) Ellipticity at 220 was measured as a function of time upon mixing mdegper 1 per mM 1 ofmM CSA; of CSA; (c) Ellipticity (c) Ellipticity at 220 at 220 was was measured measured as as a functiona function of of time time upon upon mixing mixing CSAD CSADCSAD (0.33 (0.33 μM) μM) with with CSA CSA (0. 06(0.–061 –mM)1 mM) in 20in 20mM mM potassium potassium phosphate phosphate buffer, buffer, pH pH 7.2, 7.2, containing containing (0.33 µM) with CSA (0.06–1 mM) in 20 mM potassium phosphate buffer, pH 7.2, containing 0.2 mM 0.20.2 mM mM DTT, DTT, 0.1 0.1 mM mM EDTA EDTA, and, and 0.06 0.06 mM mM PLP. PLP. The The continuous continuous line line through through the the experimental experimental DTT, 0.1 mM EDTA, and 0.06 mM PLP. The continuous line through the experimental points is the pointspoints is theis the result result of leastof least squares squares fitting fitting of dataof data to theto the Michaelis Michaelis–Menten–Menten equation, equation, which which yielded yielded result of least squares fitting of data to−1 the Michaelis–Menten equation, which yielded estimated k estimatedestimated kcat k andcat and KM K valuesM values of 5.6of 5.6 ± 0.2 ± 0.2 s sand−1 and 0.2 0.2 ± 0.02 ± 0.02 mM, mM, respectively. respectively. cat −1 and KM values of 5.6 ± 0.2 s and 0.2 ± 0.02 mM, respectively. 3.3.3.3. Decarboxylation Decarboxylation of Lof- AspartateL-Aspartate and and L- GlutamateL-Glutamate 3.3. Decarboxylation of L-Aspartate and L-Glutamate CSADCSAD-cataly-catalyzedzed decarboxylation decarboxylation of of L -aspartate L-aspartate was was observed observed by by thin thin-layer-layer chro- chro- CSAD-catalyzed decarboxylation of L-aspartate was observed by thin-layer chro- matographymatography analysis analysis after after a prolonged a prolonged incubation incubation of ofa 10 a 10mM mM so lutionsolution of ofthis this amino amino acid acid matography analysis after a prolonged incubation of a 10 mM solution of this amino acid withwith 30 30μM μ Mof ofCSAD, CSAD, at at20 20°C °C for for 16 16h (Figh (Figureure 5A). 5A). Decarboxylation Decarboxylation of ofL- glutamateL-glutamate could could with 30 µM of CSAD, at 20 ◦C for 16 h (Figure5a). Decarboxylation of L-glutamate could insteadinstead be be monitored monitored using using the the GABase GABase assay, assay, as asexplained explained in inthe the Material Material and and Methods Methods instead be monitored using the GABase assay, as explained in the Material and Methods section.section. Using Using this this assay, assay, 30 30μ Mμ M of of CSAD CSAD was was incubated incubated with with 50 50 mM mM sodium sodium section. Using this assay, 30 µM of CSAD was incubated with 50 mM sodium L-glutamate L-glutamateL-glutamate at at20 20°C, °C, and and the the product product GABA GABA was was measured measured at attime time intervals intervals (Fig (Figureure 5B). 5B). at 20 ◦C, and the product GABA was measured at time intervals (Figure5b). These results TheseThese results results clearly clearly show show that that CSAD CSAD is ableis able to todecarboxylate decarboxylate L- glutamate,L-glutamate, with with a rate a rate of of clearly show that CSAD is able to decarboxylate L-glutamate, with a rate of 0.003 µM·min−1 0.0030.003 μM·min μM·min−1 per−1 per μM μ Mof ofCSAD, CSAD, but but is alsois also able able to toslowly slowly dec decarboxylatearboxylate L- aspartate.L-aspartate. per µM of CSAD, but is also able to slowly decarboxylate L-aspartate.

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Figure 5. Decarboxylation of L-aspartate and L-glutamate. (a) Thin-layer chromatography of reaction Figure 5. Decarboxylation of L-aspartate and L-glutamate. (a) Thin-layer chromatography of reac- samples obtained by incubation of L-aspartate (mM) with CSAD (30 µM) at room temperature tion samples obtained by incubation of L-aspartate (mM) with CSAD (30 μM) at room temperature overnightovernight in in 20 20 mM mM potassium potassium phosphate phosphate bu buffer,ffer, pH pH 7.2 7.2,, containing containing 0.2 0.2 mM mM DTT, DTT, 0.1 0.1 mM mM EDTA EDTA,, andand 0.08 0.08 mM mM PLP. PLP. Samples Samples of of L L-aspartate-aspartate and β--alaninealanine were runrun asas references;references; ((bb)) Determination Determination of ofGABA GABA formation formation in in the the reaction reaction of of 32 32µM μ CSADM CSAD with with 50 mM50 mM sodium sodium glutamate, glutamate, in 20 in mM 20 mM potassium po- tassiumphosphate phosphate buffer, buff pH 7.2,er, pH containing 7.2, containing 0.2 mM 0.2 DTT mM and DTT 0.1 and mM 0.1 EDTA. mM EDTA. Measurements Measurements of amounts of amountsof GABA of were GABA carried were outcarried using out the using GABase the GABase assay, as assay, described as described in Materials in Materials and Methods. and Meth- Linear ods.regression Linear regression of data gave of adata velocity gave valuea velocity of 0.09 valueµM ·ofmin 0.09−1 .μM·min−1.

4.4. Discussion Discussion HumanHuman CSAD CSAD wi withth an an N N-terminal-terminal hexahistidine hexahistidine tag tag could could be be expressed expressed very very well well in in thethe E.E. coli coli Rosetta(DE3)Rosetta(DE3) strain,strain, althoughalthough most most of of the the protein protein was was insoluble. insoluble. By By growing growing bac- bacteriateria in minimalin minimal medium medium at low at low temperature temperature we werewe were able toable purify to purify up to 3up mg to of3 solublemg of solubleenzyme enzyme per liter per of bacterialliter of bacterial culture culture (Figure 2(Fig). Theure enzyme 2). The wasenzyme active was towards active differenttowards differensubstrates,t substrates, such as CSA,such as CA, CSA, L-aspartate, CA, L-aspartate and L-glutamate, and L-glutamate(Figures (Fig3–5ure). CSADs 3–5). activityCSAD activitywith CSA with as substrateCSA as substrate was assayed was byassayed other authorsby other using authors different using methods, different such methods, as a ra- dioisotope assay that uses L-[1-14C]CSA and measures the formation of 14CO [15], and the such as a radioisotope assay that uses L-[1-14C]CSA and measures the formation2 of 14CO2 [15]measurement, and the of measurement the hypotaurine of reactionthe hypotaurine product, derivatized reaction product, with o-phthalaldehyde derivatized with and odetected-phthalaldehyde by high-performance and detected liquidby high chromatography-performance liquid [17,19 ,chromatography29]. The CD assay [17,19,29] method is. Thesimple CD andassay direct, method because is simple it does and not direct, require because any step it does after not the require decarboxylation any step after reaction. the It has been applied to other enzymes [27,28,30], but it has never been used for CSAD. decarboxylation reaction. It has been applied to other enzymes [27,28,30], but it has never By applying this method, we determined the kinetics constants of CSA decarboxylation been used for CSAD. By applying this method, we determined the kinetics constants of (Figure4 ). The KM value of 0.2 mM found for human CSAD is very similar to the KM mea- CSA decarboxylation (Figure 4). The KM value of 0.2 mM found for human CSAD is very−1 sured for the enzyme extracted from bovine brain [15]. Moreover, the kcat value of 5.6 s similar to the KM measured for the enzyme extracted from bovine brain [15]. Moreover, is similar to that observed with other human decarboxylases, such as GAD (6–7 s−1;[31]) the kcat value of 5.6 s−1 is similar to that observed with other human decarboxylases, such and DOPA decarboxylase (3–9 s−1;[32]). Our method allowed us to demonstrate that as GAD (6–7 s−1; [31]) and DOPA decarboxylase (3–9 s−1; [32]). Our method allowed us to human CSAD can also decarboxylate CA, although at a slower rate with respect to CSA demonstrate that human CSAD can also decarboxylate CA, although at a slower rate (−0.0045·mdeg min−1 vs. −0.08 mdeg min−1). This result is in line with the hypothesis with respect to CSA (−0.0045·mdeg min−1 vs. −0.08 mdeg min−1). This result is in line with that CSAD is also responsible of the decarboxylation of CA to taurine in the alternative the hypothesis that CSAD is also responsible of the decarboxylation of CA to taurine in pathway of taurine biosynthesis (Figure1;[15]). the alternative pathway of taurine biosynthesis (Figure 1; [15]). In contrast to what was previously reported in literature, namely, that CSAD is unable to decarboxylateIn contrast to L-glutamatewhat was previously [17], we reported observed in a literature, slow reaction namely also, that with CSAD L-glutamate is un- ableand L-aspartate. to decarboxylate The decarboxylation L-glutamate [17] of L-glutamate, we observed is rather a slow expected, reaction because also CSAD with Lis-glutamate highly homologous and L-aspartate. to GAD The [17 decarboxylation], which, on the of other L-glutamate hand, can is decarboxylaterather expected, CSAD be- causesubstrates, CSAD CSA is highly and CA homologous [15,31,33]. to GAD [17], which, on the other hand, can decar- boxylateThe CSAD comparison substrates, of the CSA catalytic and CA features [15,31,33] of human. CSAD with its paralogs human glutamateThe comparison decarboxylases of the (GAD65catalytic andfeatures GAD67) of human and DOPA CSAD decarboxylase with its paralogs (DDC) human might glutamatetake advantage decarboxylases of the three-dimensional (GAD65 and GAD67) structure and of DOPA CSAD, decarboxylase which has been (DDC) solved might to a takeresolution advantage of 1.6 of Åthe by three the Structural-dimensional structure Consortium of CSAD, which (SGC) has (PDB been accession solved codeto a resolution2jis). The overallof 1.6 Å structure by the Structural of human CSADGenomics is shown Consortium in Figure (SGC)6a. The (PDB enzyme accession crystallized code

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Life 2021, 11, 438 8 of 10 2jis). The overall structure of human CSAD is shown in Figure 6A. The enzyme crystal- aslized a homo-dimer as a homo-dimer with with the PLPthe PLP cofactor cofactor bound bound through through a Schiffa Schiff base to to lysine 305 305 inin bothboth monomers.monomers. AA nitratenitrate moleculemolecule isis clearlyclearly seenseen inin thethe electronelectron density,density, coordinatedcoordinated toto His431,His431, Trp415, Trp415, Gly462, Gly462 and, and Arg461; Arg461; this this nitrate molecule molecule may maystabilize stabilize the loop the containing loop con- thetaining latter the two latter amino two acids. amino The acids. active The site active cavity site is cavity formed is byformed both monomers.by both monomers. Within bothWithin active both sites, active the sites, loop the containing loop containing Cys190/His191/Tyr192 Cys190/His191/Tyr192 can be can modelled be modelled in two in alternativetwo alternative conformations. conformations. In theIn the “closed” “closed conformation,” conformation, the the His191 His191 at at the the center center ofof thisthis looploop coordinatescoordinates withwith thethe PLP.PLP. Cys356Cys356 isis alsoalso foundfound inin twotwo conformations, conformations, possiblypossibly influencedinfluenced by by the the proximity proximity of of His191 His191 (Figure (Figure6 b).6B). An An important important structural structural feature feature of of PLP-dependentPLP-dependent decarboxylasesdecarboxylases isis aa putativeputative catalyticcatalytic looploop thatthat coverscovers thethe activeactive site.site. PartPart ofof thisthis loop,loop, fromfrom residueresidue Ser331Ser331 toto Lys341,Lys341, isis unstructuredunstructured inin chainchain A,A, whilewhile inin chainchain BB itit isis modelledmodelled inin aa muchmuch moremore openopen conformationconformation whenwhen comparedcompared toto thethe structurallystructurally andand functionallyfunctionally similar similar GAD67 GAD67 (Figure (Figure6c). 6C This). This overall overall relaxed relaxed conformation conformation of the of active the maysite mayreflect reflect the absence the absence of a bound of a substrate bound substrate or product or molecule product molecule in the CSAD in the structure. CSAD Instructure. GAD67, In this GAD67, loop has this been loop proposed has been to proposed bring catalytically to bring catalytically important residues important into resi- the proximitydues into the of PLPproximity and substrate of PLP and [34]; substrate for instance, [34]; for a highly instance, conserved a highly conserved residue tyro- presentsine residue in this present loop is in responsible this loop is for responsible substrate and for reactionsubstrate specificity and reaction in human specificity DOPA in decarboxylasehuman DOPA [decarboxylase35]. [35].

FigureFigure 6.6. StructureStructure ofof CSAD.CSAD. ((aa)) OverallOverall structurestructure ofof thethe dimericdimeric formform ofof the the enzyme. enzyme. TheThe proteinprotein backbonebackbone isis depicted depicted as as cartoon. cartoon. Chain Chain A A and and chain chain B areB are shown shown in cyan in cyan and and slate, slate, respectively. respectively. The PLPThe (inPLP yellow (in yellow color) color) bound bound to the activeto the siteactive residue site Lys305,residue andLys305 the,nitrate and the are nitrate shown are as sticks.shown ( bas) Close-upsticks. (b of) Close the active-up of site the loop active comprising site loop residuescomprising 190–192. residues The 190 loop–192. was The modelled loop was in two modelled different in conformations.two different conformations. Residues Cys190, Residues His191, Cys190, Tyr192, His191, and Cys356 Tyr192 are, and shown Cys356 as sticks;are shown (c) Comparison as sticks; (c) Comparison between the putative catalytic loop of CSAD—comprising residues Ser331 to between the putative catalytic loop of CSAD—comprising residues Ser331 to Lys341—with the Lys341—with the structurally related loop of human GAD67 (PDB 2okj). The catalytically relevant structurally related loop of human GAD67 (PDB 2okj). The catalytically relevant tyrosine residues tyrosine residues present on this loop are shown as sticks. Chain A and chain B of CSAD are shown presentin cyan onand this slate, loop respectively. are shown as Chain sticks. AChain and chain A and B chainof GAD B of 67 CSAD are shown are shown in salmon in cyan and and orange. slate, respectively.For clarity, only Chain PLP A andbound chain to CSAD B of GAD is shown, 67 are yellow shown insticks. salmon GABA and bound orange. to For GAD67 clarity, is onlyshown PLP as boundpink sticks. to CSAD is shown, yellow sticks. GABA bound to GAD67 is shown as pink sticks.

TheThe novelnovel activity activity assay assay described described in in the the present present work work will wi helpll help site-directed site-directed mutage- mu- nesistagenesis investigations investigations on the on structural the structural bases of substratebases of specificity substrate and specificity catalytic and mechanism catalytic ofmechanism human cysteine of human sulfinic cysteine acid sulfinic decarboxylase. acid decarboxylase.

SupplementaryAuthor Contribution Materials:s: A.TThe., R. followingC., R.F., C are.N. available, A.B., and online M.L. atD .https://www.mdpi.com/article/10S. designed and performed the ex- .3390/life11050438/s1periments. R.F., A.T., ,R Figure.C., and S1: M SDS-PAGE.L.D.S. critically gels. Lane read, numbering analyzed and shown discussed in Figure the S1 literature is the same and shownthe results in Figure of the2a. experiments. The lanes that A.T are., R not.C., part and of M Figure.L.D.S2.a wrote are red-crossed the manuscript in Figure and S1.supervised the Author Contributions: A.T., R.C., R.F., C.N., A.B., and M.L.D.S. designed and performed the experi- ments. R.F., A.T., R.C., and M.L.D.S. critically read, analyzed and discussed the literature and the

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results of the experiments. A.T., R.C., and M.L.D.S. wrote the manuscript and supervised the project. All the authors edited the manuscript and provided valuable discussions and criticisms. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from Istituto Pasteur Italia-Fondazione Cenci Bolognetti (to R.C.) and Sapienza University of Rome (Grant Nos. C26N158EP9, C26A13BKKY, and C26A155WEE to M.L.D.S.). Data Availability Statement: The three-dimensional structure of human CSAD can be found in the PDB databank (accession number 2jis). Acknowledgments: We thank Herwig Shüler at Karolinska Institutet, Stockholm, Sweden, for sup- port with the three-dimensional structure of CSAD and comments that greatly improved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Brosnan, J.T.; Brosnan, M.E. The Sulfur-Containing Amino Acids: An Overview. J. Nutr. 2006, 136, 1636S–1640S. [CrossRef] [PubMed] 2. Huxtable, R.J. Physiological actions of taurine. Physiol. Rev. 1992, 72, 101–163. [CrossRef][PubMed] 3. Falany, C.N.; Johnson, M.R.; Barnes, S.; Diasio, R.B. and taurine conjugation of bile acids by a single enzyme. Molecular and expression of human liver bile acid CoA:amino acid N-acyltransferase. J. Biol. Chem. 1994, 269, 19375–19379. [CrossRef] 4. Marcinkiewicz, J.; Kontny, E. Taurine and inflammatory diseases. Amino Acids 2014, 46, 7–20. [CrossRef] 5. Seidel, U.; Huebbe, P.; Rimbach, G. Taurine: A Regulator of Cellular Homeostasis and Skeletal Muscle Function. Mol. Nutr. Food Res. 2019, 63, e1800569. [CrossRef] 6. Lambert, I.H. Regulation of the cellular content of the organic osmolyte taurine in mammalian cells. Neurochem. Res. 2004, 29, 27–63. [CrossRef] 7. Moran, J.; Salazar, P.; Pasantes-Morales, H. Effect of tocopherol and taurine on membrane fluidity of retinal rod outer segments. Exp. Eye Res. 1987, 45, 769–776. [CrossRef] 8. Wu, J.-Y.; Prentice, H. Role of taurine in the central nervous system. J. Biomed. Sci. 2010, 17, S1. [CrossRef] 9. Schaffer, S.W.; Shimada-Takaura, K.; Jong, C.J.; Ito, T.; Takahashi, K. Impaired energy of the taurinedeficient heart. Amino Acids 2016, 48, 549–558. [CrossRef][PubMed] 10. Hayes, K.C.; Carey, R.; Schmidt, S.Y. Retinal degeneration associated with taurine deficiency in the cat. Science 1975, 188, 949–951. [CrossRef] 11. Verner, A.M.; McGuire, W.; Craig, J.S. Effect of taurine supplementation on growth and development in preterm or low birth weight infants. Cochrane Database Syst. Rev. 2007.[CrossRef][PubMed] 12. Murakami, S. Role of taurine in the pathogenesis of obesity. Mol. Nutr. Food Res. 2015, 59, 1353–1363. [CrossRef][PubMed] 13. Chesney, R.W. Taurine: Its biological role and clinical implications. Adv. Pediatr. 1985, 32, 1–42. [PubMed] 14. Siefken, W.; Carstensen, S.; Springmann, G.; Wittern, K.-P.; Wenck, H.; Stäb, F.; Sauermann, G.; Schreiner, V.; Doering, T.; Janeke, G.; et al. Role of Taurine Accumulation in Keratinocyte Hydration. J. Investig. Dermatol. 2003, 121, 354–361. [CrossRef] 15. Wu, J.Y. Purification and characterization of cysteic acid and cysteine sulfinic acid decarboxylase and L- from bovine brain. Proc. Natl. Acad. Sci. USA 1982, 79, 4270–4274. [CrossRef] 16. Jansonius, J.N. Structure, evolution and action of B6-dependent enzymes. Curr. Opin. Struct. Biol. 1998, 8, 759–769. [CrossRef] 17. Tappaz, M.; Bitoun, M.; Reymond, I.; Sergeant, A. Characterization of the cDNA coding for rat brain cysteine sul-finate decarboxylase: Brain and liver enzymes are identical proteins encoded by two distinct mRNAs. J. Neurochem. 1999, 73, 903–912. [CrossRef] 18. Tang, X.W.; Hsu, C.C.; Schloss, J.V.; Faiman, M.D.; Wu, E.; Yang, C.Y.; Wu, J.Y. Protein and taurine biosynthesis in vivo and in vitro. J. Neurosci. 1997, 17, 6947–6951. [CrossRef] 19. Agnello, G.; Chang, L.L.; Lamb, C.M.; Georgiou, G.; Stone, E.M. Discovery of a Substrate Selectivity Motif in Amino Acid Decarboxylases Unveils a Taurine Biosynthesis Pathway in . ACS Chem. Biol. 2013, 8, 2264–2271. [CrossRef][PubMed] 20. Tevatia, R.; Allen, J.; Rudrappa, D.; White, D.; Clemente, T.E.; Cerutti, H.; Demirel, Y.; Blum, P. The taurine biosynthetic pathway of microalgae. Algal Res. 2015, 9, 21–26. [CrossRef] 21. Gill, S.C.; von Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182, 319–326. [CrossRef] 22. Tramonti, A.; De Biase, D.; Giartosio, A.; Bossa, F.; John, R.A. The Roles of His-167 and His-275 in the Reaction Catalyzed by Glutamate Decarboxylase from . J. Biol. Chem. 1998, 273, 1939–1945. [CrossRef][PubMed] 23. Harris, C.M.; Johnson, R.J.; Metzler, D.E. Band-shape analysis and resolution of electronic spectra of and other 3-hydroxypyridine-4-. Biochim. Biophys. Acta Gen. Subj. 1976, 421, 181–194. [CrossRef] Life 2021, 11, 438 10 of 10

24. Hayashi, H.; Mizuguchi, H.; Kagamiyama, H. Rat liver aromatic L-amino acid decarboxylase: Spectroscopic and kinetic analysis of the coenzyme and reaction intermediates. 1993, 32, 812–818. [CrossRef][PubMed] 25. Hayashi, H.; Tsukiyama, F.; Ishii, S.; Mizuguchi, H.; Kagamiyama, H. Acid-base chemistry of the reaction of ar-omatic L-amino acid decarboxylase and dopa analyzed by transient and steady-state kinetics: Preferential binding of the substrate with its amino group unprotonated. Biochemistry 1999, 38, 15615–15622. [CrossRef] 26. Voltattorni, C.B.; Minelli, A.; Vecchini, P.; Fiori, A.; Turano, C. Purification and characterization of 3,4-dihydroxyphenylalanine decarboxyase from pig kidney. Eur. J. Biochem. 1979, 93, 181–188. [CrossRef] 27. Brooks, H.B.; Phillips, M.A. Circular Dichroism Assay for Decarboxylation of Optically Pure Amino Acids: Application to Ornithine Decarboxylase. Anal. Biochem. 1996, 238, 191–194. [CrossRef] 28. Fogle, E.J.; Toney, M.D. Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates. Biochim. Biophys. Acta Proteins Proteom. 2011, 1814, 1113–1119. [CrossRef][PubMed] 29. Winge, I.; Teigen, K.; Fossbakk, A.; Mahootchi, E.; Kleppe, R.; Sköldberg, F.; Kämpe, O.; Haavik, J. Mammalian CSAD and GADL1 have distinct biochemical properties and patterns of brain expression. Neurochem. Int. 2015, 90, 173–184. [CrossRef] 30. Noda, M.; Matoba, Y.; Kumagai, T.; Sugiyama, M. A novel assay method for an amino acid racemase reaction based on circular dichroism. Biochem. J. 2005, 389, 491–496. [CrossRef] 31. Blindermann, J.-M.; Maitre, M.; Ossola, L.; Mandel, P. Purification and Some Properties of l-Glutamate Decarboxylase from Human Brain. J. Biol. Inorg. Chem. 1978, 86, 143–152. [CrossRef][PubMed] 32. Siaterli, M.Z.; Vassilacopoulou, D.; Fragoulis, E.G. Cloning and expression of human placental L-Dopa decar-boxylase. Neurochem. Res. 2003, 28, 797–803. [CrossRef][PubMed] 33. Oertel, W.H.; Schmechel, D.E.; Weise, V.K.; Ransom, D.H.; Tappaz, M.L.; Krutzsch, H.C.; Kopin, I.J. Comparison of cysteine sulphinic acid decarboxylase isoenzymes and decarboxylase in rat liver and brain. Neuroscience 1981, 6, 2701–2714. [CrossRef] 34. Fenalti, G.; Law, R.H.P.; Buckle, A.M.; Langendorf, C.; Tuck, K.; Rosado, C.J.; Faux, N.G.; Mahmood, K.; Hampe, C.S.; Banga, J.P.; et al. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat. Struct. Mol. Biol. 2007, 14, 280–286. [CrossRef][PubMed] 35. Bertoldi, M.; Gonsalvi, M.; Contestabile, R.; Voltattorni, C.B. of Tyrosine 332 to Converts Dopa Decar- boxylase into a Decarboxylation-dependent Oxidative Deaminase. J. Biol. Chem. 2002, 277, 36357–36362. [CrossRef]