Insights Into the Mn Binding Site in the Agmatinase-Like Protein (ALP): A

International Journal of Molecular Sciences

Article Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals

María-Belen Reyes 1, José Martínez-Oyanedel 1,*, Camila Navarrete 1, Erika Mardones 1, Ignacio Martínez 1,Mónica Salas 2, Vasthi López 3, María García-Robles 4, Estefania Tarifeño-Saldivia 1, Maximiliano Figueroa 1, David García 1 and Elena Uribe 1,*

1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4070386, Chile; [email protected] (M.-B.R.); [email protected] (C.N.); [email protected] (E.M.); [email protected] (I.M.); [email protected] (E.T.-S.); [email protected] (M.F.); [email protected] (D.G.) 2 Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia 5110566, Chile; [email protected] 3 Departamento de Ciencias Biomédicas, Universidad Católica del Norte, Coquimbo 1781421, Chile; [email protected] 4 Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 3349001, Chile; [email protected] * Correspondence: [email protected] (J.M.-O.); [email protected] (E.U.)

Received: 28 April 2020; Accepted: 5 June 2020; Published: 10 June 2020

Abstract: Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the “central-ALP”, which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km 2+ for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

Keywords: ureohydrolase; ALP; manganese

1. Introduction Agmatine (1-amino-4-guanidinobutane) results from the decarboxylation of L-arginine by arginine decarboxylase (ADC) and is hydrolyzed to putrescine and urea by agmatinase (AGM) or agmatinase-like protein (ALP), shown in Figure1A. Agmatine has been directly associated with many important cellular functions, such as the modulation of insulin release from pancreatic cells [1–3],

Int. J. Mol. Sci. 2020, 21, 4132; doi:10.3390/ijms21114132 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4132 2 of 11 renal sodium excretion [4,5], inhibition of nitric oxide synthase [6,7], neuroprotective effects [8], increased tolerance to morphine [9], modulation of ethanol anxiolysis [10], and regulation of polyamine biosynthesis [11,12]. It is considered a neurotransmitter/neuromodulator [13] because it regulates the release of catecholamines and potentiates opioid analgesia [14]. Indeed, the injection of agmatine produces anticonvulsant, antineurotoxic and antidepressant-like actions in animals [13,15]. It has also been linked to other central nervous system disorders. Specifically, preclinical studies have demonstrated the beneficial effects of agmatine administration on diseases such as depression, anxiety, Int.hypoxic J. Mol. Sci. ischemia, 2019, 20, nociception,x FOR PEER REVIEW morphine v3 of 12 tolerance, memory, Parkinson’s disease, Alzheimer’s disease, traumatic brain injury-related disorders, and epilepsy [16,17]. The various biological processes on which indicateagmatine that, is involved in contrast suggest with thatAGM it mightand ARG, require histidine the fine residues regulation are not of its critical cellular for concentrations. the catalytic activity Thus, ofunderstanding ALP. its synthesis (by ADC) and hydrolysis (by AGM or ALP) is crucial to understanding its regulation.In the present study, we identified the active site region of ALP and proposed the residues required for Mn2+ binding.

Figure 1. (A) Pathway of agmatine biosynthesis and breakdown. ADC: arginine decarboxylase; ALP: Figureagmatinase-like 1. (A) Pathway protein; of AGM: agmatine agmatinase. biosynthesis (B) Schematic and breakdown illustration. ADC: of thearginine Mn2+ decarboxylase;binding site of E.ALP: coli 2+ agmatinase-likeAGM. The enzyme protein; can accommodateAGM: agmatinase. two closely(B) Schematic spaced illustration Mn2+ ions inof theirthe Mn active binding sites, usingsite ofhighly E. coli AGM. The enzyme can accommodate two closely spaced Mn2+ ions in their active sites, using highly conserved amino acid side chains [18]. conserved amino acid side chains [18]. AGM belongs to the ureohydrolases enzyme family, which requires bivalent ions for its catalytic 2.activity, Results especially and Discussion Mn2+. On the active site, six strictly conserved amino acid residues are responsible for metal coordination; four Asp and two His residues [18–20], as shown in Figure1B. While AGM 2.1. Manganese Binding Site in ALP from Escherichia coli has been extensively studied, a detailed characterization of mammalian AGM is still lacking.Using comparative In this sense, modeling our laboratory we generated has identified a structural a rat brainmodel protein of ∆LIM-ALP, with significant including agmatinase the Mn2+ bindingactivity insite, vitro but [without21,22]. Interestingly,considering the the first deduced 30 residues amino and acid 3 longer sequence loops of this(H67 enzyme to G111, greatly E145 to di S178,ffers andfrom E345 all knownto P417) members that represent of the the ureohydrolase principal sequen family,ce singularity lacking the of characteristic this protein. MnThe2 +modelligands passed and thecatalytic stereochemistry residues [ 21and]. Basedenergy on conformation its agmatinase assessm activityent, andas described the lack in of sequenceSection 4.2. conservation, The model presentswe referred the togeneral this enzyme folding asof “agmatinase-likethis protein family, protein” presenting (ALP). only differences in the length of some of the secondaryALP, which structure is present elements. only in As mammals shown andin Figure has been 2, the identified amino acids in rat present brain tissues in the (astrocytes putative Mn and2+ binding site of ALP contain numerous variations regarding the conserved1 amino acid in the neurons of hypothalamus and hippocampus [23]), display a kcat of 0.9 0.2 s− for agmatine hydrolysis ureohydrolase family. In general, in metalloproteins, metal ions are ±coordinated by donor groups as and a Km value of 3.0 0.2 mM for agmatine [21,22]. Furthermore, we demonstrated its ability to generate ± nitrogen,putrescine oxygen, under inor vivosulfurconditions centers belonging [22,24]. Fromto the these amino results, acid residues and given of thatthe protein. human-AGM There are does three not 2+ “majordisplay binders” agmatinase of Mn activity ions: [25 oxygen,26], ALP atoms might from be carboxyl the enzyme groups regulating of aspartic agmatine and glutamic concentrations acids side in chains,mammals and and, imidazole therefore, nitrogen the various atoms from important histidine functions side chain. associated Minor withbinders agmatine are: oxygen [16,18 atoms]. from hydroxyl groups of serine and threonine side chains; amide nitrogen and oxygen atoms from asparagine A singular component of ALP protein sequence is the motif C-X16-H-X2-C-X2-C-X2-C-X21-C-X2-C and glutamine side chains; sulfur atoms from thiol group of cysteine and thioether group of methionine (X denotes any amino acid) in the C-terminus (residues 459–510). This motif is characteristic of the and oxygen atoms from peptide bonds of all the amino acids, including even hydrophobic ones [37]. so-called LIM-domain, commonly found in mammalian proteins and involved in protein–protein We suggest that in ALP the Mn2+ interactions involve a new type of ligation between E190, N213, Q215, D217, E288, and K290 and the Mn2+ ions; similar residue types interact with Mn2+ ions in others proteins [38]. While aspartate is often found stabilizing binuclear metal centers, residues such as glutamate and asparagine can also play this role [35]. For example, it has been reported that Asn81 stabilizes the binuclear Mn2+ center of metallophosphoesterase from a marine bacteria [39], and Asn233 plays a similar role in the binuclear Zn2+ center of the betalactamase of Bacillus cereus [40]. Furthermore, glutamic residues (Glu235 and Glu204) have been described as stabilizing the binuclear Co2+ center of a

Int. J. Mol. Sci. 2020, 21, 4132 3 of 11 interactions [27,28]. We have shown that a deletion mutant of ALP, lacking the LIM-domain, is catalytically more active than the wild-type ALP; the truncated variant (∆LIM-ALP) exhibits a 10-fold higher kcat and a three-fold lower Km value for agmatine [22,26]. In this study, ∆LIM-ALP is used as a positive control in the characterization of ALP variants. Regarding the metal ion requirements in ALP agmatinase activity, we have determined that ALP requires Mn2+ ions for its activity, and the presence of EDTA produces a total inactivation, which is reverted by the addition of metal ions [21,29]. In this respect, ALP behaves similar to all Mn2+-dependent members of the ureohydrolase family, such as the E. coli AGM [30,31] and arginases (ARG) [32–34]. In their fully active state, these enzymes contain a binuclear Mn2+ center, which, according to our results, is also present in ALP [29,35,36]. Due to the lack of structural information and the low degree of sequence identity between ALP and all known ureohydrolases, the active site in ALP is completely unknown. As we mentioned earlier, in ureohydrolases, aspartate and histidine amino acids are ligands for the metallic cofactor. However, mutations of the five His residues in ALP did not generate significant changes except for the mutant H206A, which produced a 10-fold decreased affinity for Mn2+ binding [29]. These results indicate that, in contrast with AGM and ARG, histidine residues are not critical for the catalytic activity of ALP. In the present study, we identified the active site region of ALP and proposed the residues required for Mn2+ binding.

2. Results and Discussion

2.1. Manganese Binding Site in ALP Using comparative modeling we generated a structural model of ∆LIM-ALP, including the Mn2+ binding site, but without considering the first 30 residues and 3 longer loops (H67 to G111, E145 to S178, and E345 to P417) that represent the principal sequence singularity of this protein. The model passed the stereochemistry and energy conformation assessment, as described in Section 4.2. The model presents the general folding of this protein family, presenting only differences in the length of some of the secondary structure elements. As shown in Figure2, the amino acids present in the putative Mn2+ binding site of ALP contain numerous variations regarding the conserved amino acid in the ureohydrolase family. In general, in metalloproteins, metal ions are coordinated by donor groups as nitrogen, oxygen, or sulfur centers belonging to the amino acid residues of the protein. There are three “major binders” of Mn2+ ions: oxygen atoms from carboxyl groups of aspartic and glutamic acids side chains, and imidazole nitrogen atoms from histidine side chain. Minor binders are: oxygen atoms from hydroxyl groups of serine and threonine side chains; amide nitrogen and oxygen atoms from asparagine and glutamine side chains; sulfur atoms from thiol group of cysteine and thioether group of methionine and oxygen atoms from peptide bonds of all the amino acids, including even hydrophobic ones [37]. We suggest that in ALP the Mn2+ interactions involve a new type of ligation between E190, N213, Q215, D217, E288, and K290 and the Mn2+ ions; similar residue types interact with Mn2+ ions in others proteins [38]. While aspartate is often found stabilizing binuclear metal centers, residues such as glutamate and asparagine can also play this role [35]. For example, it has been reported that Asn81 stabilizes the binuclear Mn2+ center of metallophosphoesterase from a marine bacteria [39], and Asn233 plays a similar role in the binuclear Zn2+ center of the betalactamase of Bacillus cereus [40]. Furthermore, glutamic residues (Glu235 and Glu204) have been described as stabilizing the binuclear Co2+ center of a methionine aminopeptidase from E. coli [35] and the binuclear Mn2+ center (Glu 56-57-58) of a pyrophosphohydrolase from E. coli [41]. On the other hand, Gln and Lys residues have not been described with such a role, however, Gln displays similar physicochemical properties to Asn, and Lys has been linked to the second coordination sphere interactions of metal ions [42]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v4 of 12 methionine aminopeptidase from E. coli [35] and the binuclear Mn2+ center (Glu 56-57-58) of a Int. J. Mol. Sci. 2020, 21, 4132 4 of 11 pyrophosphohydrolase from E. coli [41]. On the other hand, Gln and Lys residues have not been described with such a role, however, Gln displays similar physicochemical properties to Asn, and Lys has beenBased linked on our to the structural second comparativecoordination modelsphere andinteractions the literature of metal supporting ions [42]. the stabilizing role of the residuesBased on identified, our structural we suggest comparative that in model ALP, the and Mn the2+ literatureinteractions supporting are performed the stabilizing by residues role whichof the areresidues not the identified, classic Asp we andsuggestHis foundthat in inALP, the the ureohydrolases Mn2+ interactions enzyme are familyperformed [18]. by residues which are not the classic Asp and His found in the ureohydrolases enzyme family [18].

2+ Figure 2. Model of thethe putativeputative MnMn2+ binding site for ∆∆LIM-ALP, generatedgenerated throughthrough thethe softwaresoftware 2+ MODELLER 9.22. 9.22. The The Mn Mn2+ bindingbinding site site proposed proposed including including Asn213, Asn213, Gln215, Gln215, Asp217, Asp217, Glu288, Glu288, and Lys290,and Lys290, instead instead of four of Asp four andAsp twoand His two (theHis scheme(the scheme does not does include not include Glu 190). Glu 190). 2.2. Expression and Characterization of Central-ALP 2.2. Expression and Characterization of Central-ALP To study the putative Mn2+ binding site and define the region containing the active site of ALP, To study the putative Mn2+ binding site and define the region containing the active site of ALP, we we focused our analysis on 210 amino acid regions of ALP (from T140–S350) flanking the putative focused our analysis on 210 amino acid regions of ALP (from T140–S350) flanking the putative Mn2+ 2+ Mnbindingbinding site, as site,shown as shownin Figure in 3 Figure (complete3 (complete sequence sequence of ALP is of in ALP Supplementary is in Supplementary Material). Materials).This region Thiswas selected region was to minimize selected to the minimize disruption the of disruption the secondary of the structure secondary predicted structure by predicted our model, by ourand model,it was andcalled it the was central-ALP called the central-ALP variant due to variant its location due to in its the location WT-ALP, in theas shown WT-ALP, in Figure as shown 3A,B. in The Figure sequence3A,B. Theof central-ALP sequence ofwas central-ALP amplified by was PCR amplified and cloned by PCRon the and H6pQE60 cloned E. on coli the expression H6pQE60 vector,E. coli whichexpression adds vector,a His-Tag which for addsfurther a Hispurification.-Tag for further The expressed purification. and Thepartially expressed purified and enzyme partially was purified confirmed enzyme by wasWestern confirmed blot, as bypreviously Western described blot, as previously by Mella et describedal. [23] and, by as Mella expected, et al. the [23 central-ALP] and, as expected, variant had the central-ALPa molecular weight variant of had 25 KDa, a molecular as shown weight in Figure of 25 3C. KDa, Further, as shown we performed in Figure3 C.agmatinase Further, weactivity performed assays 2+ agmatinasefor central-ALP activity in the assays presence for central-ALP and absence in theof Mn presence2+. As shown and absence in Figure of Mn 4, our. As results shown indicate in Figure that4, 2+ ourcentral-ALP results indicate (grey bars) that did central-ALP not show (greyactivity bars) in the did absence not show of activityMn2+, while in the its absence activity ofincreased Mn , while two- itsfold activity when the increased metal ion two-fold was added when to the the metal media. ion We was used added ∆LIM-ALP to the media. as a positive We used control∆LIM-ALP (white bars as a positivein Figure control 4), this (white variant bars displays in Figure high4), AGM this variant activity displays (10-fold highhigher AGM than activity ALP) and (10-fold its purification higher than is ALP)simple and [22]. its As purification expected, similar is simple results [22]. were As expected,observed with similar ∆LIM-ALP results were increasing observed its activity with ∆ LIM-ALPeight-fold increasingin the presence its activity of Mn2+ eight-fold. These results in the showed presence that of central-ALP Mn2+. These can results hydrolyze showed agmatine that central-ALP and its activity can hydrolyzeis Mn2+ dependent. agmatine and its activity is Mn2+ dependent. 2+2+ We also studied AGMAGM activityactivity whenwhen thethe variantsvariants werewere heatedheated atat 6565◦ °CC ininpresence presence of of Mn Mn , cooledcooled 2+ 2+ down to room room temperature, temperature, and and AGM AGM activity activity was was measured measured at at37 37°C◦ (5C (5mM mM Mn Mnon theon media). the media). We Weobserved observed that that central-ALP central-ALP increased increased its AGM its AGM activity activity four-fold, four-fold, while while ∆LIM-ALP∆LIM-ALP variant variant displayed displayed 20- 20-foldfold increased increased activity. activity. These These results results agree agree with with our our previous observations wherewhere wild-type-ALPwild-type-ALP increased its its activity activity ~4-fold ~4-fold [29] [29. ].The The increased increased activity activity of ALP, of ALP, produced produced for the for heating the heating at 65 at°C 65 with◦C withMn2+, Mnis a2 +typical, is a typicalcharacteristic characteristic of ureohydrolases of ureohydrolases,, such as the such rat asliver the arginase rat liver [18,39] arginase and [Helicobacter18,39] and Helicobacterpylori ARG [32]. pylori It ARGhas been [32]. suggested It has been that suggested the heating that ofthe the heatingenzymeof in thethe enzymepresence in of the Mn presence2+ increases of Mn 2+ increases the activity of the enzyme through the stabilization of its binuclear Mn2+ center [32]. These results together suggest that an Mn2+ binuclear center is formed on the central-ALP variant and that it contains enough residues to stabilize the Mn2+ ions. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v5 of 12 theInt. J.activity Mol. Sci. 2019of the, 20 , enzymex FOR PEER through REVIEW thev5 of stabilization 12 of its binuclear Mn2+ center [32]. These results together suggest that an Mn2+ binuclear center is formed on the central-ALP variant and that it contains enoughthe activity residues of the to stabilizeenzyme thethrough Mn2+ ions.the stabilization of its binuclear Mn2+ center [32]. These results Int.together J. Mol. suggest Sci. 2020, 21that, 4132 an Mn2+ binuclear center is formed on the central-ALP variant and that it contains5 of 11 enough residues to stabilize the Mn2+ ions.

Figure 3. (A). Comparative scheme of ALP, ∆LIM-ALP, and central-ALP. The regions shown in blue are identicalFigure 3. between(A). Comparative the different scheme proteins. of ALP, (B).∆ LIM-ALP,Alignment andof central-ALP central-ALP. construction The regions shownand ALP, in bluethe proposedFigureare identical 3. metal(A). betweenComparative ligands are the indicated dischemeﬀerent of in proteins. ALP, red. (∆CLIM-ALP,). (WesternB). Alignment and blot central-ALP. analysis of central-ALP of central-ALP The regions construction obtained shown in from and blue ALP,N are2+- NTAidenticalthe proposed chromatography. between metal the ligands Adifferent band are of indicatedproteins. approximately ( inB). red. Alignment 35 (C kDa). Western corresponding of central-ALP blot analysis to central-ALPconstruction of central-ALP wasand observed. obtainedALP, the Anproposedfrom anti-ALP N2+ -NTAmetal antibody ligands chromatography. di arelution indicated 1:2000 A band inwas red. used. of (C approximately). Western blot 35 analysis kDa corresponding of central-ALP to obtained central-ALP from wasN2+- NTAobserved. chromatography. An anti-ALP A antibody band of dilutionapproximately 1:2000 was35 kDa used. corresponding to central-ALP was observed. An anti-ALP antibody dilution 1:2000 was used.

Figure 4. Effect of Mn2+ on the agmatinase activity of central-ALP and ∆LIM-ALP. Empty bars indicate Figure∆LIM-ALP 4. Effect activity of Mn and2+ on black the agmatinase bars indicate activity central-ALP of central-ALP activity; and -Mn ∆LIM-ALP2+ indicates. Empty the measurementbars indicate ∆ofLIM-ALP activity activity in the absence and black of addedbars indicate manganese; central-ALP+Mn2+ activity;indicates -Mn the2+ measurementindicates the measurement of activity with of 2+ 2+ activityFigure5 mM MnCl4.in Effectthe2 .absence In of addition,Mn of on added the activity agmatinase manganese; was measured activity +Mn of withindicates central-ALP previous the measurement treatmentand ∆LIM-ALP of incubationof. activityEmpty barswith at 65 indicate 5◦ CmM for 2+ 2+ MnCl∆5LIM-ALP min,2. In in addition, the activity presence activityand andblack was absence bars measured indicate of 5mM with central-ALP Mn previous, then treatmentactivity; the tubes - Mnof were incubation indicates cooled at andthe 65 measurement°C the for agmatinase 5 min, inof theactivity presence wasin the determinedand absence absence ofat ofadded 37 5 mMC. manganese; Negative Mn2+, then control +theMn 2+tubes ( indicates), measured were cooledthe withmeasurement and only the 80 agmatinase mM of activity agmatine activity with in 50 5 was mMmM ◦ − determinedMnClglycine–NaOH2. In addition, at 37 (pH°C. Negativeactivity 9.0) and was control 5 mM measured MnCl (−), measured2 .with previous with only treatment 80 mM agmatineof incubation in 50 at mM 65 glycine–NaOH°C for 5 min, in 2+ (pHthe 9.0)presence and 5 and mM absence MnCl2. of 5 mM Mn , then the tubes were cooled and the agmatinase activity was determinedTo study the at 37 kinetics °C. Negative of ALP control activation (−), measured by Mn 2with+, we only pre-incubated 80 mM agmatine central-ALP in 50 mM glycine–NaOH with 5 mM Mn 2+ at 65To(pH◦C study during9.0) and the 5 dikinetics mMfferent MnCl of periods,2 .ALP activation as shown by in Mn Figure2+, we5 pre-incubatedA. We found thatcentral-ALP central-ALP with progressively5 mM Mn2+ at 65increases °C during its activity different until periods, rising toas ashown plateau, in and Figure a similar 5A. We tendency found was that observed central-ALP for ALP progressively [29]. Then, we measuredTo study the AGM kinetics activity of ALP at di activationfferent concentrations by Mn2+, we pre-incubated of Mn2+ to determine central-ALP an activation with 5 mM constant Mn2+ at 2+ (65K act°CMn during), as different shown in periods, Figure5 asB andshown Table in1 .Fi Wegure used 5A. central-ALP We found inthat two central-ALP conditions, progressively previously 2+ heated at 65 ◦C in the presence of Mn and without pre-heating. In both cases, the constant was

similar to ∆LIM-ALP, as shown in Table1. This Kact has been directly associated with the dissociation 2+ 2+ constant (Kd) of the enzyme–Mn complex [18]. Therefore, we suggest that the aﬃnity for Mn is maintained in central-ALP and the ligands required to coordinate the metal ions are present on this variant. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v6 of 12 increases its activity until rising to a plateau, and a similar tendency was observed for ALP [29]. Then, we measured AGM activity at different concentrations of Mn2+ to determine an activation constant (Kact Mn2+), as shown in Figure 5B and Table 1. We used central-ALP in two conditions, previously heated at 65 °C in the presence of Mn2+ and without pre-heating. In both cases, the constant was similar to ΔLIM- ALP, as shown in Table 1. This Kact has been directly associated with the dissociation constant (Kd) of the Int. J. Mol. Sci. 2020, 21, 4132 6 of 11 enzyme–Mn2+ complex [18]. Therefore, we suggest that the affinity for Mn2+ is maintained in central- ALP and the ligands required to coordinate the metal ions are present on this variant.

Figure 5. A 2+ Figure 5. ((A).) Enzymatic activation assays of of central-ALP central-ALP with with Mn Mn2+. The. The incubations incubations were were carried carried out out at 2+ at65°C 65 ◦atC different at diﬀerent times, times, in the in presence the presence of Mn of2+ Mn 5mM,5 then mM, the then tubes the were tubes co wereoled cooledand agmatinase and agmatinase activity 2+ activitywas determined was determined at 37 °C. at 37(B)◦ C.Determination (B) Determination of the ofactivation the activation constant constant by Mn by2+ Mn in central-ALPin central-ALP.. The 2+ Theenzyme enzyme was wasincubated incubated for 15 for min 15 minat 37 at °C 37 with◦C with varying varying concentrations concentrations of Mn of2+ Mn in 10in mM 10 mMTris–HCl Tris–HCl (pH (pH8.5), 8.5),50 mM 50 KCl, mM and KCl, 10 and mM 10 nitril mMotriacetic nitrilotriacetic acid as a acid metal as ion a metal buffer. ion Then, buﬀ er.agmatine Then, was agmatine added wasand addedincubated and at incubated 37 °C for 15 at 37min.◦C Then, for 15 the min. AGM Then, activities the AGM wereactivities determined. were The determined. studies were The performed studies were performed in duplicates and repeated twice. Activation constants (K ) were estimated from the in duplicates and repeated twice. Activation constants (Ka) were estimateda from the hyperbolic 2+ 2+ 2+ 2+K hyperbolicdependence dependence of agmatinase of agmatinase activity on activityfree-Mn on concentrations free-Mn concentrations (similarly to (similarly a Km for Mn to a ). m for Mn ). Table 1. Kinetic parameters of agmatine hydrolysis of ALP variants. The kinetic characterization of central-ALP showed a Michaelis–Menten saturation curve, shown 2+ 1 2+ in Figure 6, in both conditions, previouslyK heatedm (mM) with Mn atk cat65 (s°C− )and withoutKact Mn heating.(M) The Km of central-ALPALP-wt was 1.8 mM (for heated assay) 3.0 and0.20 1.2 mM (without 0.9 heating),0.2 which1.88 is similar10 8 to the Km of ± ± × − ΔLIM-ALP∆LIM-ALP (1.2 mM). As seen in Table 1, the 1.2 kcat 0.04of central-ALP decreased 10 1 by half 3.6when10 compared8 to the ± ± × − wild-type-ALP.∆LIM-ALP Therefore,/D217A central-ALP displays No activity similar Km but No activitydiffers in catalytic No activity efficiency. These results suggest∆LIM-ALP that/ E287Acentral-ALP/K289A might bindNo to activity the substrate as it No does activity to ALP-WT, No however, activity its catalysis might require∆LIM-ALP residues/N213A outside/Q215A the/D217A centralNo region activity to be similarly No efficient. activity No activity 8 Central-ALP 1.2 0.37 0.4 0.5 1.7 10− ± ± × 8 Central-ALP (65 ◦C) 1.2 0.37 0.8 0.4 2.2 10 Table 1. Kinetic parameters± of agmatine hydrolysis± of ALP variants. × −

The kinetic characterization of central-ALP showedKm (mM) a Michaelis–Menten kcat (s−¹) saturationKact curve,Mn2+ (M shown) 2+ in FigureALP-wt6, in both conditions, previously heated with 3.0 ± Mn 0.20 at 65 ◦C 0.9 and ± 0.2 without heating. 1.88 × The10−8 Km of central-ALP was 1.8 mM (for heated assay) and 1.2 mM (without heating), which is similar to the Km of ∆LIM-ALP 1.2 ± 0.04 10 ± 1 3.6 × 10−8 ∆LIM-ALP (1.2 mM). As seen in Table1, the kcat of central-ALP decreased by half when compared to∆ theLIM-ALP/D217A wild-type-ALP. Therefore, central-ALP displays No activity similar Km Nobut activity differs in catalytic No activity efficiency. These results suggest that central-ALP might bind to the substrate as it does to ALP-WT, however, Int.∆ LIM-ALP/E287A/K289AJ. Mol. Sci. 2019, 20, x FOR PEER REVIEW v7 of 12 No activity No activity No activity its catalysis might require residues outside the central region to be similarly efficient. ∆LIM-ALP/N213A/Q215A/D217A No activity No activity No activity

Central-ALP 1.2 ± 0.37 0.4 ± 0.5 1.7 × 10−8

Central-ALP (65°C) 1.2 ± 0.37 0.8 ± 0.4 2.2 × 10−8

Figure 6. Michaelis–Menten plot for central-ALP. The saturation curve for central-ALP previously

heatedFigure to 6. 65Michaelis–Menten◦ C for 5 min is observed plot for central-ALP in white circles. The and saturation in black curve circles for without central-ALP heating. previously The activities heated wereto 65 performed ° C for 5 min at37 is ◦observedC and at in pH white 9.0 with circles MnCl and2 5in mM black and circles diﬀerent without agmatine heating. concentrations. The activities were performed at 37 °C and at pH 9.0 with MnCl2 5 mM and different agmatine concentrations.

2.3. Site-Directed Mutagenesis of Putative Mn2+ Ligands.

Finally, we performed a functional analysis of the putative Mn2+ ligands in ALP, identified in the present study. To do so, we generated a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of ∆LIM-ALP. These variants were expressed and identified in chromatography fractions by means of Western blot using a specific anti-ALP antibody, shown in Figure 7 [23,24]. As shown in Table 1, we did not observe agmatinase activity in these ALP mutants. The results indicate that these residues are required for the ALP activity. These findings are in agreement with observations performed for ureohydrolases, such as the rat and human arginase, and the E. coli agmatinase. For those enzymes, the mutation of one residue coordinating Mn2+ causes partial or total loss of the enzymatic activity [43,44]. For example, on E. coli agmatinase, the mutation of the ligand H126N reduced agmatinase activity by 50%, while the mutation H151N produced a total loss of activity [44].

Figure 7. Western blot identification of mutated ∆LIM-ALP variants. Using Western blot using an anti- ALP antibody (dilution 1: 2000), a band of approximately 70 kD was detected corresponding to the wild- type (control), the double mutant, and triple mutant of ∆LIM-ALP.

3. Conclusions In the present work, we conclude that the active site of the ALP enzyme resides in the central-region (from T140–S350). This region contains the ligands necessary for Mn2+ binding and catalysis. The fact that ALP is a ureohydrolase, is Mn2+-dependent, and displays such sequence divergence suggests that we are studying a new type of ureohydrolase with a new type of Mn2+ ligand. Our model proposes new residues for the Mn2+ binding site and the mutants’ results indicate the importance in the agmatinase

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v7 of 12

Int. J.Figure Mol. Sci. 6.2020 Michaelis–Menten, 21, 4132 plot for central-ALP. The saturation curve for central-ALP previously heated7 of 11 to 65 ° C for 5 min is observed in white circles and in black circles without heating. The activities were

performed at 37 °C and at pH 9.0 with MnCl2 5 mM and different agmatine concentrations. 2.3. Site-Directed Mutagenesis of Putative Mn2+ Ligands 2.3. Site-DirectedFinally, we Mutagenesis performed of a functionalPutative Mn analysis2+ Ligands. of the putative Mn2+ ligands in ALP, identified in the presentFinally, study.we performed To do so, a wefunctional generated analysis a simple of the mutant putative D217A, Mn2+ aligands double in mutant ALP, identified E288A/K290A, in the presentand a triple study. mutant To do N213A so, we/Q215A generated/D217A a simple of ∆LIM-ALP. mutant D217A, These variantsa double were mutant expressed E288A/K290A, and identified and a triplein chromatography mutant N213A/Q215A/D217A fractions by means of ∆ ofLIM-ALP. Western blotThese using variants a specific were anti-ALP expressed antibody, and identified shown in chromatographyFigure7[ 23,24]. fractions As shown by in means Table of1, Western we did not blot observe using a agmatinasespecific anti-ALP activity antibody, in these shown ALP mutants.in Figure 7The [23,24]. results As indicate shown in that Table these 1, residueswe did not are observe required agmatinase for the ALP activity activity. in Thesethese ALP findings mutants. are in The agreement results indicatewith observations that these performedresidues are for required ureohydrolases, for the ALP such activity. as the rat These and findings human arginase, are in agreement and the E. with coli observationsagmatinase. performed For those enzymes,for ureohydrolases, the mutation such of oneas the residue rat and coordinating human arginase, Mn2+ causes and the partial E. coli or agmatinase.total loss of For the those enzymatic enzymes, activity the [mutation43,44]. For of example,one residue on coordinatingE. coli agmatinase, Mn2+ causes the mutation partial or of total the lossligand of H126Nthe enzymatic reduced activity agmatinase [43,44]. activity For example, by 50%, whileon E. thecoli mutation agmatinase, H151N the producedmutation of a total the ligand loss of H126Nactivity reduced [44]. agmatinase activity by 50%, while the mutation H151N produced a total loss of activity [44].

FigureFigure 7. 7. WesternWestern blot blot identification identiﬁcation of ofmutated mutated ∆LIM-ALP∆LIM-ALP variants. variants. Using Using Western Western blot using blot using an anti- an ALPanti-ALP antibody antibody (dilution (dilution 1: 2000), 1:2000), a band a bandof approxim of approximatelyately 70 kD 70 was kD detected was detected corresponding corresponding to the towild- the typewild-type (control), (control), the double the double mutant, mutant, and triple and mutant triple mutant of ∆LIM-ALP. of ∆LIM-ALP.

3. Conclusions Conclusions InIn the the present present work, work, we we conclude conclude that that the the active active si sitete of of the the ALP ALP enzyme resides in the central-region 2+ (from T140–S350). This region containscontains the ligandsligands necessarynecessary forfor MnMn2+ binding and catalysis. The fact 2+ thatthat ALP is a ureohydrolase, isis MnMn2+-dependent,-dependent, and and displays displays such such se sequencequence divergence suggests that 2+ we are studying a new typetype ofof ureohydrolaseureohydrolase withwith aa newnew typetype ofof MnMn2+ ligand. Our Our model model proposes proposes new new 2+ residues for the Mn2+ bindingbinding site site and and the the mutants’ mutants’ results results in indicatedicate the importance in the agmatinase activity in ALP. Finally, the crystal structure of ALP is required to validate our model and support our 2+ findings concerning Mn binding. Considering that ALP has emerged as a central enzyme in regulating crucial neurological processes, a detailed understanding of its interaction with Mn2+ and how its activity is controlled will be essential in defining this enzyme as a promising drug target to treat human afflictions [35,45].

4. Materials and Methods

4.1. Materials Agmatine, glycine, Tris, SDS, and all other reagents were of the highest quality commercially available (most from Sigma Aldrich Chemical Co. Louis, MO, USA). Restriction enzymes, as well as enzymes and reagents for PCR, were obtained from Invitrogen Co. (Carlsbad, CA, USA). The synthetic nucleotide primers were obtained from the Fermelo Biotec Co. (Santiago, Chile).

4.2. Molecular Modeling For the putative Mn2+ binding site, a molecular model for ∆LIM-ALP was generated through the software MODELLER 9.22 [46]. Because the searching for templates through Blastp server (https: //blast.ncbi.nlm.nih.gov/) and threading servers, such as Genthreader (http://bioinf.cs.ucl.ac.uk/psipred/) and Phyre2 (http://www.sbg.bio.ic.ac.uk/~{}phyre2), did not give any suitable results, we selected members of the ureohydrolase family available in the PDB server (https://www.rcsb.org/): Deinococcus Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v8 of 12

activity in ALP. Finally, the crystal structure of ALP is required to validate our model and support our findings concerning Mn2+ binding. Considering that ALP has emerged as a central enzyme in regulating crucial neurological processes, a detailed understanding of its interaction with Mn2+ and how its activity is controlled will be essential in defining this enzyme as a promising drug target to treat human afflictions [35,45].

4. Materials and Methods

4.2. Molecular Modeling Int. J. Mol. Sci. 2020, 21, 4132 8 of 11 radiodurans (PDB id: 1WOH, 22% identity, 33% similarity, 1.75 Ǻ resolution), ClostridiumClostridium didifficilefficile (PDB(PDB id: 3LHL, 22% identity, 32% similarity, 2.3 Ǻ resolution), id: 3LHL, 22% identity, 32% similarity, 2.3 resolution), BurkholderiaBurkholderia thailandensisthailandensis (PDB(PDB id:id: 4DZ4,4DZ4, 22%22% identity, 31% similarity, 1.7 Ǻ resolution) all agmatinases and a guanidine butyrase from Pseudomonas aeruginosa (PDB id: 3NIO, 22% identity, 33% similarity, identity, 31% similarity, 1.7 resolution) all agmatinases and2.0 Ǻ a guanidineresolution), butyrase and a proclavaminate from Pseudomonas amidino hydrolase from Streptomyces clavuligerus (PDB id: aeruginosa (PDB id: 3NIO, 22% identity, 33% similarity, 2.0 1GQ6,resolution), 22% identity, and a proclavaminate 33% similarity, amidino1,75 Ǻ resolution), several of them including Mn2+. The alignment of the templates was done by Clustal using BLOSUM45 matrix, and was improved by structural hydrolase from Streptomyces clavuligerus (PDB id: 1GQ6, 22% identity, 33% similarity, 1,75 resolution), alignment to shift the gaps to zones free of secondary structure elements. Finally 30 models were several of them including Mn2+. The alignment of the templates was done by Clustal using BLOSUM45 construct and matrix, and was improved by structural alignment to shift the gaps to zones free of secondary structure elements. Finally 30 models were construct and assessed by DOPE. The final model was evaluated with Procheck (https://servicesn.mbi.ucla.edu/PROCHECK/) for stereochemistry and Prosa Server (https://prosa.services.came.sbg.ac.at/) for energetic assessment.assessed The residuesby DOPE. forming theThe site werefinal model was evaluated with Procheck 2+ proposed through structural alignment with the templates(https://servicesn.mbi.ucla.edu/PROCHECK/) that included Mn in the structure. for stereochemistry and Prosa Server (https://prosa.services.came.sbg.ac.at/) for energetic assessment. The residues forming the site were 4.3. Enzyme Preparations proposed through structural alignment with the templates that included Mn2+ in the structure. The sequence of central-ALP was amplified using the PCR technique (with Kod, Merck, high fidelity DNA-polymerase) from the plasmid H6pQE60-29.2,4.3. containing Enzyme Preparations the ALP cDNA as the template. The desired sequence was confirmed by automated DNA sequenceThe sequence analysis. of The central-ALP amplified fragmentwas amplified using the PCR technique (with Kod, Merck, high of 630 bp (210 aa), was directionally cloned into the histidine-taggedfidelity DNA-polymerase) pQE60 bacterial from the expression plasmid H6pQE60-29.2, containing the ALP cDNA as the template. vector, and the histidine-tagged proteins were expressedThe in E. desired coli strain sequence JM109, was following confirmed induction by automated DNA sequence analysis. The amplified fragment of with 0.5 mM isopropyl-β-D-thiogalactopyranoside. The630 central-ALPbp (210 aa), was was directionally partially purified cloned byinto the histidine-tagged pQE60 bacterial expression vector, means of DEAE-cellulose anion exchange chromatographyand (calibrated the histidine-tagged with Tris-HCl proteins 10 mM, were pH expressed 7.5), in E. coli strain JM109, following induction with 0.5 eluted with KCl 250 mM and an NTA–Ni2+ affinity chromatography.mM isopropyl- Theβ-D-thiogalactopyranoside. purity of all preparations The central-ALP was partially purified by means of DEAE- was ~70%. The single mutant D217A, the double mutantcellulose E288A anion/K290A, exchange and chromatography the triple mutant, (calibrated with Tris-HCl 10 mM, pH 7.5), eluted with KCl N213A/Q215A/K290A of the putative metal-ligand site of ALP250 weremM and obtained an NTA–Ni by using2+ affinity the QuikChange chromatography.® The purity of all preparations was ~70%. The single Site-Directed Mutagenesis Kit (Stratagene) with the plasmidmutant H6pQE60-29.2, D217A, the containing double mutant the ∆LIM-ALP E288A/K290A, and the triple mutant, N213A/Q215A/K290A of the cDNA as the template. The presence of the desired mutationputative and metal-ligand the absence ofsite unwanted of ALP we changesre obtained by using the QuikChange® Site-Directed Mutagenesis were confirmed by automated DNA sequence analysis. Kit (Stratagene) with the plasmid H6pQE60-29.2, containing the ∆LIM-ALP cDNA as the template. The presence of the desired mutation and the absence of unwanted changes were confirmed by automated 4.4. ALP Activity Determination DNA sequence analysis. Routinely, the ALP activities were determined by measuring the formation of urea (product) using 80 mM agmatine in 50 mM glycine–NaOH (pH 9.0) and 5 mM MnCl2. All the assays were initiated by adding the enzyme to the substrate, buffer, and MnCl2 solution, which were previously equilibrated at 37 ◦C. The urea was determined by the formation of a colored complex with α-isonitrosopropiophenone [33], measuring the absorbance at 540 nm. Initial velocity studies were performed in duplicates and repeated three times. Kinetic parameters were obtained by fitting the experimental data to the appropriate Michaellis–Menten equation (vi = VmaxS/Km + S) by using nonlinear regression with Graph Pad Prism version 7.0 for Windows (Graph Pad Software Inc., San Diego, CA, USA). Protein concentration was determined using the standard Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as standard.

4.5. Enzyme–Metal Interactions Analysis

2+ For reactivation assays with Mn , the enzyme was incubated for 15 min at 37 ◦C with varying concentrations of Mn2+ in 10 mM Tris–HCl (pH 8.5), 50 mM KCl, and 10 mM nitrilotriacetic acid as a metal ion buﬀer. Then, agmatine was added and incubated at 30 ◦C for 15 min and the AGM activities were determined. The studies were performed in duplicates and repeated twice. Activation 2+ constants (Ka) were estimated from the hyperbolic dependence of agmatinase activity on free-Mn concentrations, using nonlinear regression analysis in Graph Pad Prism 5.0 (similar to a Km for Mn2+). Free Mn2+ concentrations were calculated using a dissociation constant of 3.98 10 8 M and × − Int. J. Mol. Sci. 2020, 21, 4132 9 of 11 a pKa value of 9.8 for nitrilotriacetic acid (NTA) using the software MaxChelator WINMAXC 2.4 (http://www.stanford.edu/~{}cpatton/maxc.html)[36].

4.6. Statistical Analysis The results were evaluated with GraphPad Prism v7.0 using an analysis of variance (ANOVA), multiple comparison tests, or an unpaired two-tailed t-test.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/11/ 4132/s1. Author Contributions: M.-B.R.: Conceptualization Ideas, Methodology. J.M.-O.: Conceptualization Ideas, Methodology, Visualization Preparation, Writing—Reviewing and Editing. C.N.: Methodology. E.M.: Methodology. I.M.: Methodology. M.S.: Resources, Funding Acquisition. V.L.: Resources, Funding Acquisition. M.G.-R.: Resources, Funding Acquisition. E.T.-S.: Writing—Reviewing and Editing. M.F.: Writing- Reviewing and Editing. D.G.: Conceptualization Ideas, Writing—viewing and Editing. E.U.: Conceptualization Ideas, Methodology, Investigation, Writing—riginal Draft, Writing—eview & Editing. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a grant from VRID-Enlace 217.037.022-1, University of Concepción-Chile. Acknowledgments: Special thanks to the Dean of the Faculty of Biological Sciences, Dra. Soraya Gutiérrez and to the VRID of the University of Concepción, for the support received in the publication of this manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Chan, S.L.F. Clonidine-displacing substance and its putative role in control of insulin secretion: A minireview. Gen. Pharmacol. Vasc. Syst. 1998, 31, 525–529. [CrossRef] 2. Su, C.-H.; Liu, I.-M.; Chung, H.-H.; Cheng, J.-T. Activation of I2-imidazoline receptors by agmatine improved insulin sensitivity through two mechanisms in type-2 diabetic rats. Neurosci. Lett. 2009, 457, 125–128. [CrossRef][PubMed] 3. Lee, J.-P.; Chen, W.; Wu, H.-T.; Lin, K.-C.; Cheng, J.-T. Metformin can activate imidazoline I-2 Receptors to lower plasma glucose in type 1-like diabetic rats. Horm. Metab. Res. 2011, 43, 26–30. [CrossRef][PubMed] 4. Penner, B.; Smyth, D.D. Natriuresis following central and peripheral administration of agmatine in the rat. Pharmacology 1996, 53, 160–169. [CrossRef][PubMed] 5. Satriano, J.; Cunard, R.; Peterson, O.W.; Dousa, T.; Gabbai, F.B.; Blantz, R.C. Effects on kidney filtration rate by agmatine requires activation of ryanodine channels for nitric oxide generation. Am. J. Physiol. Ren. Physiol. 2008, 294, F795–F800. [CrossRef][PubMed] 6. Qiu, W.-W.; Zheng, R.-Y. Neuroprotective effects of receptor imidazoline 2 and its endogenous ligand agmatine. Neurosci. Bull. 2006, 22, 187–191. [PubMed] 7. Mun, C.H.; Lee, W.T.; Park, K.A.; Lee, J.E. Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain. Anat. Cell Biol. 2010, 43, 230–240. [CrossRef][PubMed] 8. Lee, W.T.; Hong, S.; Yoon, S.H.; Kim, J.H.; Park, K.A.; Seong, G.J.; Lee, J.E. Neuroprotective effects of agmatine on oxygen-glucose deprived primary-cultured astrocytes and nuclear translocation of nuclear factor-kappa B. Brain Res. 2009, 1281, 64–70. [CrossRef][PubMed] 9. Li, F.; Wu, N.; Su, R.; Chen, Y.; Lu, X.; Liu, Y.; Li, J. Imidazoline receptor antisera-selected/Nischarin regulates the effect of agmatine on the development of morphine dependence. Addict. Biol. 2012, 17, 392–408. [CrossRef][PubMed] 10. Taksande, B.G.; Kotagale, N.R.; Patel, M.R.; Shelkar, G.P.; Ugale, R.R.; Chopde, C.T. Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats. Eur. J. Pharmacol. 2010, 637, 89–101. [CrossRef][PubMed] 11. Satriano, J.; Matsufuji, S.; Murakami, Y.; Lortie, M.J.; Schwartz, D.; Kelly, C.J.; Hayashi, S.; Blantz, R.C. Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J. Biol. Chem. 1998, 273, 15313–15316. [CrossRef][PubMed] 12. Vargiu, C.; Cabella, C.; Belliardo, S.; Cravanzola, C.; Grillo, M.A.; Colombatto, S. Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase. Eur. J. Biochem. 1999, 259, 933–938. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 4132 10 of 11

13. Piletz, J.E.; Aricioglu, F.; Cheng, J.-T.; Fairbanks, C.A.; Gilad, V.H.; Haenisch, B.; Halaris, A.; Hong, S.; Lee, J.E.; Li, J.; et al. Agmatine: Clinical applications after 100 years in translation. Drug Discov. Today 2013, 18, 880–893. [CrossRef][PubMed] 14. Wu, N.; Su, R.-B.; Li, J. Agmatine and imidazoline receptors: Their role in opioid analgesia, tolerance and dependence. Cell. Mol. Neurobiol. 2008, 28, 629–641. [CrossRef][PubMed] 15. Dixit, M.P.; Thakre, P.P.; Pannase, A.S.; Aglawe, M.M.; Taksande, B.G.; Kotagale, N.R. Imidazoline binding sites mediates anticompulsive-like effect of agmatine in marble-burying behavior in mice. Eur. J. Pharmacol. 2014, 732, 26–31. [CrossRef][PubMed] 16. Barua, S.; Kim, J.Y.; Kim, J.Y.; Kim, J.H.; Lee, J.E. Therapeutic Effect of Agmatine on Neurological Disease: Focus on Ion Channels and Receptors. Neurochem. Res. 2019, 44, 735–750. [CrossRef][PubMed] 17. Moretti, M.; Matheus, F.C.; de Oliveira, P.A.; Neis, V.B.; Ben, J.; Walz, R.; Rodrigues, A.L.S.; Prediger, R.D. Role of agmatine in neurodegenerative diseases and epilepsy. Front. Biosci. 2014, 6, 341–359. [CrossRef] 18. Uribe, E.; Reyes, M.-B.; Martínez, I.; Mella, K.; Salas, M.; Tarifeño-Saldivia, E.; López, V.; García-Robles, M.; Martínez-Oyanedel, J.; Figueroa, M.; et al. Functional analysis of the Mn2+ requirement in the catalysis of ureohydrolases arginase and agmatinase—A historical perspective. J. Inorg. Biochem. 2020, 202, 110812. [CrossRef] 19. Scolnick, L.R.; Kanyo, Z.F.; Cavalli, R.C.; Ash, D.E.; Christianson, D.W. Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function. Biochemistry 1997, 36, 10558–10565. [CrossRef][PubMed] 20. Ash, D.E. Structure and function of arginases. J. Nutr. 2004, 134, 2760S–2764S. [CrossRef][PubMed] 21. Uribe, E.; Salas, M.; Enríquez, S.; Orellana, M.S.; Carvajal, N. Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family. Arch. Biochem. Biophys. 2007, 461, 146–150. [CrossRef][PubMed] 22. Castro, V.; Fuentealba, P.; Henríquez, A.; Vallejos, A.; Benítez, J.; Lobos, M.; Díaz, B.; Carvajal, N.; Uribe, E. Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein. Arch. Biochem. Biophys. 2011, 512, 107–110. [CrossRef][PubMed] 23. Mella, C.; Martínez, F.; de Los Angeles García, M.; Nualart, F.; Castro, V.; Bustos, P.; Carvajal, N.; Uribe, E. Expression and localization of an agmatinase-like protein in the rat brain. Histochem. Cell Biol. 2010, 134, 137–144. [CrossRef][PubMed] 24. Romero, N.; Benítez, J.; Garcia, D.; González, A.; Bennun, L.; García-Robles, M.A.; López, V.; Wilson, L.A.; Schenk, G.; Carvajal, N.; et al. Mammalian agmatinases constitute unusual members in the family of Mn2+-dependent ureahydrolases. J. Inorg. Biochem. 2017, 166, 122–125. [CrossRef][PubMed] 25. Iyer, R.K.; Kim, H.K.; Tsoa, R.W.; Grody, W.W.; Cederbaum, S.D. Cloning and characterization of human agmatinase. Mol. Genet. Metab. 2002, 75, 209–218. [CrossRef][PubMed] 26. García, D.; Ordenes, P.; Benítez, J.; González, A.; García-Robles, M.A.; López, V.; Carvajal, N.; Uribe, E. Cloning of two LIMCH1 isoforms: Characterization of their distribution in rat brain and their agmatinase activity. Histochem. Cell Biol. 2016, 145, 305–313. [CrossRef][PubMed] 27. Feuerstein, R.; Wang, X.; Song, D.; Cooke, N.E.; Liebhaber, S.A. The LIM/double zinc-finger motif functions as a protein dimerization domain. Proc. Natl. Acad. Sci. USA 1994, 91, 10655–10659. [CrossRef][PubMed] 28. Kadrmas, J.L.; Beckerle, M.C. The LIM domain: From the cytoskeleton to the nucleus. Nat. Rev. Mol. Cell Biol. 2004, 5, 920–931. [CrossRef][PubMed] 29. Quiñones, M.; Cofre, J.; Benítez, J.; García, D.; Romero, N.; González, A.; Carvajal, N.; García, M.; López, V.; Schenk, G.; et al. Insight on the interaction of an agmatinase-like protein with Mn2+ activator ions. J. Inorg. Biochem. 2015, 145, 65–69. [CrossRef][PubMed] 30. Carvajal, N.; López, V.; Salas, M.; Uribe, E.; Herrera, P.; Cerpa, J. Manganese is essential for catalytic activity ofescherichia coliagmatinase. Biochem. Biophys. Res. Commun. 1999, 258, 808–811. [CrossRef][PubMed] 31. Carvajal, N.; Orellana, M.S.; Salas, M.; Enríquez, P.; Alarcón, R.; Uribe, E.; López, V. Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group. Arch. Biochem. Biophys. 2004, 430, 185–190. [CrossRef][PubMed] 32. Cama, E.; Colleluori, D.M.; Emig, F.A.; Shin, H.; Kim, S.W.; Kim, N.N.; Traish, A.M.; Ash, D.E.; Christianson, D.W. Human arginase II: Crystal structure and physiological role in male and female sexual arousal. Biochemistry 2003, 42, 8445–8451. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 4132 11 of 11

33. Alarcón, R.; Orellana, M.S.; Neira, B.; Uribe, E.; García, J.R.; Carvajal, N. Mutational analysis of substrate recognition by human arginase type I—Agmatinase activity of the N130D variant. Febs J. 2006, 273, 5625–5631. [CrossRef][PubMed] 34. Dutta, A.; Mazumder, M.; Alam, M.; Gourinath, S.; Sau, A.K. Metal-induced change in catalytic loop positioning in Helicobacter pylori arginase alters catalytic function. Biochem. J. 2019, 476, 3595–3614. [CrossRef][PubMed] 35. Mitić,N.; Miraula, M.; Selleck, C.; Hadler, K.S.; Uribe, E.; Pedroso, M.M.; Schenk, G. Chapter three—Catalytic mechanisms of metallohydrolases containing two metal ions. In Advances in Protein Chemistry and Structural Biology; Metal-Containing Enzymes; Christov, C.Z., Ed.; Academic Press: New York, NY, USA, 2014; Volume 97, pp. 49–81. 36. Cofre, J.; Montes, P.; Vallejos, A.; Benítez, J.; García, D.; Martínez-Oyanedel, J.; Carvajal, N.; Uribe, E. Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein. J. Inorg. Biochem. 2014, 132, 92–95. [CrossRef][PubMed] 37. Harding, M.M. The architecture of metal coordination groups in proteins. Acta Crystallogr. D Biol Crystallogr. 2004, 60, 849–859. [CrossRef][PubMed] 38. Khrustaleva, T.A. Secondary structure preferences of Mn2+ binding sites in bacterial proteins. Adv. Bioinform. 2014, 2014, 501841. [CrossRef][PubMed] 39. Wei, T.; Quareshy, M.; Zhang, Y.; Scanlan, D.; Chen, Y. Manganese Is essential for PlcP metallophosphoesterase activity involved in lipid remodeling in abundant marine heterotrophic bacteria. Appl. Environ. Microbiol. 2018, 84, 1109–1118. [CrossRef][PubMed] 40. Phelan, E.K.; Miraula, M.; Selleck, C.; Ollis, D.L.; Schenk, G.; Mitić,N. Metallo-β-lactamases: A major threat to human health. Am. J. Mol. Biol. 2014, 4, 89–104. [CrossRef] 41. Harris, T.; Wu, G.; Massiah, M.; Mildvan, A. Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase. Biochemistry 2000, 39, 1655–1674. [CrossRef][PubMed] 42. Dudev, T.; Lin, Y.; Dudev, M.; Lim, C. First second shell interactions in metal binding sites in proteins: − A PDB survey and DFT/CDM calculations. J. Am. Chem. Soc. 2003, 125, 3168–3180. [CrossRef][PubMed] 43. Orellana, M.S.; López, V.; Uribe, E.; Fuentes, M.; Salas, M.; Carvajal, N. Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies. Arch. Biochem Biophys 2002, 403, 155–159. [CrossRef] 44. Salas, M.; López, V.; Uribe, E.; Carvajal, N. Studies on the interaction of Escherichia coli agmatinase with manganese ions: Structural and kinetic studies of the H126N and H151N variants. J. Inorg. Biochem. 2004, 98, 1032–1036. [CrossRef][PubMed] 45. Benítez, J.; García, D.; Romero, N.; González, A.; Martínez-Oyanedel, J.; Figueroa, M.; Salas, M.; López, V.; García-Robles, M.; Dodd, P.R.; et al. Metabolic strategies for the degradation of the neuromodulator agmatine in mammals. Metabolism 2018, 81, 35–44. [CrossRef][PubMed] 46. Canutescu, A.A.; Shelenkov, A.A.; Dunbrack, R.L. A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci. 2003, 12, 2001–2014. [CrossRef][PubMed]

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