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

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 for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ 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.


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 Figure 1A. Agmatine has been directly associated with many important cellular functions, such as the modulation of insulin release from pancreatic cells [1][2][3], The enzyme can accommodate two closely spaced Mn 2+ ions in their active sites, using highly conserved amino acid side chains [18].

Manganese Binding Site in ALP
Using comparative modeling we generated a structural model of ∆LIM-ALP, including the Mn 2+ 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 Figure 2, the amino acids present in the putative Mn 2+ 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 Mn 2+ 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 Mn 2+ interactions involve a new type of ligation between E190, N213, Q215, D217, E288, and K290 and the Mn 2+ ions; similar residue types interact with Mn 2+ 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 Mn 2+ center of metallophosphoesterase from a marine bacteria [39], and Asn233 plays a similar role in the binuclear Zn 2+ center of the betalactamase of Bacillus cereus [40]. Furthermore, The enzyme can accommodate two closely spaced Mn 2+ ions in their active sites, using highly conserved amino acid side chains [18]. AGM belongs to the ureohydrolases enzyme family, which requires bivalent ions for its catalytic activity, especially Mn 2+ . On the active site, six strictly conserved amino acid residues are responsible for metal coordination; four Asp and two His residues [18][19][20], as shown in Figure 1B. While AGM from Escherichia coli has been extensively studied, a detailed characterization of mammalian AGM is still lacking. In this sense, our laboratory has identified a rat brain protein with significant agmatinase activity in vitro [21,22]. Interestingly, the deduced amino acid sequence of this enzyme greatly differs from all known members of the ureohydrolase family, lacking the characteristic Mn 2+ ligands and catalytic residues [21]. Based on its agmatinase activity and the lack of sequence conservation, we referred to this enzyme as "agmatinase-like protein" (ALP).
ALP, which is present only in mammals and has been identified in rat brain tissues (astrocytes and neurons of hypothalamus and hippocampus [23]), display a k cat of 0.9 ± 0.2 s −1 for agmatine hydrolysis and a K m value of 3.0 ± 0.2 mM for agmatine [21,22]. Furthermore, we demonstrated its ability to generate putrescine under in vivo conditions [22,24]. From these results, and given that human-AGM does not display agmatinase activity [25,26], ALP might be the enzyme regulating agmatine concentrations in mammals and, therefore, the various important functions associated with agmatine [16,18].
A singular component of ALP protein sequence is the motif C-X16-H-X2-C-X2-C-X2-C-X21-C-X2-C (X denotes any amino acid) in the C-terminus (residues 459-510). This motif is characteristic of the so-called LIM-domain, commonly found in mammalian proteins and involved in protein-protein 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 k cat and a three-fold lower K m 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 Mn 2+ 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 Mn 2+ -dependent members of the ureohydrolase family, such as the E. coli AGM [30,31] and arginases (ARG) [32][33][34]. In their fully active state, these enzymes contain a binuclear Mn 2+ 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 Mn 2+ 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 Mn 2+ binding.

Manganese Binding Site in ALP
Using comparative modeling we generated a structural model of ∆LIM-ALP, including the Mn 2+ 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 Figure 2, the amino acids present in the putative Mn 2+ 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 Mn 2+ 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 Mn 2+ interactions involve a new type of ligation between E190, N213, Q215, D217, E288, and K290 and the Mn 2+ ions; similar residue types interact with Mn 2+ 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 Mn 2+ center of metallophosphoesterase from a marine bacteria [39], and Asn233 plays a similar role in the binuclear Zn 2+ center of the betalactamase of Bacillus cereus [40]. Furthermore, glutamic residues (Glu235 and Glu204) have been described as stabilizing the binuclear Co 2+ center of a methionine aminopeptidase from E. coli [35] and the binuclear Mn 2+ 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].
Based on our structural comparative model and the literature supporting the stabilizing role of the residues identified, we suggest that in ALP, the Mn 2+ interactions are performed by residues which are not the classic Asp and His found in the ureohydrolases enzyme family [18].
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW v4 of 12 methionine aminopeptidase from E. coli [35] and the binuclear Mn 2+ 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].
Based on our structural comparative model and the literature supporting the stabilizing role of the residues identified, we suggest that in ALP, the Mn 2+ interactions are performed by residues which are not the classic Asp and His found in the ureohydrolases enzyme family [18].

Expression and Characterization of Central-ALP
To study the putative Mn 2+ binding site and define the region containing the active site of ALP, we focused our analysis on 210 amino acid regions of ALP (from T140-S350) flanking the putative Mn 2+ binding site, as shown in Figure 3 (complete sequence of ALP is in Supplementary Material). This region was selected to minimize the disruption of the secondary structure predicted by our model, and it was called the central-ALP variant due to its location in the WT-ALP, as shown in Figure 3A,B. The sequence of central-ALP was amplified by PCR and cloned on the H6pQE60 E. coli expression vector, which adds a His-Tag for further purification. The expressed and partially purified enzyme was confirmed by Western blot, as previously described by Mella et al. [23] and, as expected, the central-ALP variant had a molecular weight of 25 KDa, as shown in Figure 3C. Further, we performed agmatinase activity assays for central-ALP in the presence and absence of Mn 2+ . As shown in Figure 4, our results indicate that central-ALP (grey bars) did not show activity in the absence of Mn 2+ , while its activity increased twofold when the metal ion was added to the media. We used ∆LIM-ALP as a positive control (white bars in Figure 4), this variant displays high AGM activity (10-fold higher than ALP) and its purification is simple [22]. As expected, similar results were observed with ∆LIM-ALP increasing its activity eight-fold in the presence of Mn 2+ . These results showed that central-ALP can hydrolyze agmatine and its activity is Mn 2+ dependent.
We also studied AGM activity when the variants were heated at 65 °C in presence of Mn 2+ , cooled down to room temperature, and AGM activity was measured at 37 °C (5 mM Mn 2+ on the media). We observed that central-ALP increased its AGM activity four-fold, while ∆LIM-ALP variant displayed 20fold increased activity. These results agree with our previous observations where wild-type-ALP increased its activity ~4-fold [29]. The increased activity of ALP, produced for the heating at 65 °C with Mn 2+ , is a typical characteristic of ureohydrolases, such as the rat liver arginase [18,39] and Helicobacter pylori ARG [32]. It has been suggested that the heating of the enzyme in the presence of Mn 2+ increases

Expression and Characterization of Central-ALP
To study the putative Mn 2+ binding site and define the region containing the active site of ALP, we focused our analysis on 210 amino acid regions of ALP (from T140-S350) flanking the putative Mn 2+ binding site, as shown in Figure 3 (complete sequence of ALP is in Supplementary Materials). This region was selected to minimize the disruption of the secondary structure predicted by our model, and it was called the central-ALP variant due to its location in the WT-ALP, as shown in Figure 3A,B. The sequence of central-ALP was amplified by PCR and cloned on the H6pQE60 E. coli expression vector, which adds a His-Tag for further purification. The expressed and partially purified enzyme was confirmed by Western blot, as previously described by Mella et al. [23] and, as expected, the central-ALP variant had a molecular weight of 25 KDa, as shown in Figure 3C. Further, we performed agmatinase activity assays for central-ALP in the presence and absence of Mn 2+ . As shown in Figure 4, our results indicate that central-ALP (grey bars) did not show activity in the absence of Mn 2+ , while its activity increased two-fold when the metal ion was added to the media. We used ∆LIM-ALP as a positive control (white bars in Figure 4), this variant displays high AGM activity (10-fold higher than ALP) and its purification is simple [22]. As expected, similar results were observed with ∆LIM-ALP increasing its activity eight-fold in the presence of Mn 2+ . These results showed that central-ALP can hydrolyze agmatine and its activity is Mn 2+ dependent.
We also studied AGM activity when the variants were heated at 65 • C in presence of Mn 2+ , cooled down to room temperature, and AGM activity was measured at 37 • C (5 mM Mn 2+ on the media). We observed that central-ALP increased its AGM activity four-fold, while ∆LIM-ALP variant displayed 20-fold increased activity. These results agree with our previous observations where wild-type-ALP increased its activity~4-fold [29]. The increased activity of ALP, produced for the heating at 65 • C with Mn 2+ , is a typical characteristic of ureohydrolases, such as the rat liver arginase [18,39] and Helicobacter pylori ARG [32]. It has been suggested that the heating of the enzyme in the presence of Mn 2+ increases the activity of the enzyme through the stabilization of its binuclear Mn 2+ center [32]. These results together suggest that an Mn 2+ binuclear center is formed on the central-ALP variant and that it contains enough residues to stabilize the Mn 2+ ions.
the activity of the enzyme through the stabilization of its binuclear Mn 2+ center [32]. These results together suggest that an Mn 2+ binuclear center is formed on the central-ALP variant and that it contains enough residues to stabilize the Mn 2+ ions.  To study the kinetics of ALP activation by Mn 2+ , we pre-incubated central-ALP with 5 mM Mn 2+ at 65 °C during different periods, as shown in Figure 5A. We found that central-ALP progressively the activity of the enzyme through the stabilization of its binuclear Mn 2+ center [32]. These results together suggest that an Mn 2+ binuclear center is formed on the central-ALP variant and that it contains enough residues to stabilize the Mn 2+ ions.  To study the kinetics of ALP activation by Mn 2+ , we pre-incubated central-ALP with 5 mM Mn 2+ at 65 °C during different periods, as shown in Figure 5A. We found that central-ALP progressively To study the kinetics of ALP activation by Mn 2+ , we pre-incubated central-ALP with 5 mM Mn 2+ at 65 • C during different periods, as shown in Figure 5A. We found that central-ALP progressively 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 Mn 2+ to determine an activation constant (K act Mn 2+ ), as shown in Figure 5B and Table 1. We used central-ALP in two conditions, previously heated at 65 • C in the presence of Mn 2+ and without pre-heating. In both cases, the constant was similar to ∆LIM-ALP, as shown in Table 1. This K act has been directly associated with the dissociation constant (K d ) of the enzyme-Mn 2+ complex [18]. Therefore, we suggest that the affinity for Mn 2+ is maintained in central-ALP and the ligands required to coordinate the metal ions are present on this variant. 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 Mn 2+ to determine an activation constant (Kact Mn 2+ ), as shown in Figure 5B and Table 1. We used central-ALP in two conditions, previously heated at 65 °C in the presence of Mn 2+ 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 enzyme-Mn 2+ complex [18]. Therefore, we suggest that the affinity for Mn 2+ is maintained in central-ALP and the ligands required to coordinate the metal ions are present on this variant. The kinetic characterization of central-ALP showed a Michaelis-Menten saturation curve, shown in Figure 6, in both conditions, previously heated with Mn 2+ at 65 °C and without heating. The Km of central-ALP was 1.8 mM (for heated assay) and 1.2 mM (without heating), which is similar to the Km of ΔLIM-ALP (1.2 mM). As seen in Table 1, the kcat of central-ALP decreased by half when compared to the wild-type-ALP. Therefore, central-ALP displays similar Km but differs in catalytic efficiency. These results suggest that central-ALP might bind to the substrate as it does to ALP-WT, however, its catalysis might require residues outside the central region to be similarly efficient.   The kinetic characterization of central-ALP showed a Michaelis-Menten saturation curve, shown in Figure 6, in both conditions, previously heated with Mn 2+ at 65 • C and without heating. The K m of central-ALP was 1.8 mM (for heated assay) and 1.2 mM (without heating), which is similar to the K m of ∆LIM-ALP (1.2 mM). As seen in Table 1, the k cat of central-ALP decreased by half when compared to the wild-type-ALP. Therefore, central-ALP displays similar K m but differs in catalytic efficiency. These results suggest that central-ALP might bind to the substrate as it does to ALP-WT, however, its catalysis might require residues outside the central region to be similarly efficient.

Site-Directed Mutagenesis of Putative Mn 2+ Ligands.
Finally, we performed a functional analysis of the putative Mn 2+ ligands in ALP, identified in the present study. To do so, we generated a simple mutant D217A, a double mutant E288A/K290A, and a

Site-Directed Mutagenesis of Putative Mn 2+ Ligands
Finally, we performed a functional analysis of the putative Mn 2+ 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 Mn 2+ 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].

Site-Directed Mutagenesis of Putative Mn 2+ Ligands.
Finally, we performed a functional analysis of the putative Mn 2+ 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 Mn 2+ 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].

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 Mn 2+ binding and catalysis. The fact that ALP is a ureohydrolase, is Mn 2+ -dependent, and displays such sequence divergence suggests that we are studying a new type of ureohydrolase with a new type of Mn 2+ ligand. Our model proposes new residues for the Mn 2+ binding site and the mutants' results indicate the importance in the agmatinase

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 Mn 2+ binding and catalysis. The fact that ALP is a ureohydrolase, is Mn 2+ -dependent, and displays such sequence divergence suggests that we are studying a new type of ureohydrolase with a new type of Mn 2+ ligand. Our model proposes new residues for the Mn 2+ binding site and the mutants' results indicate the importance in the agmatinase activity in ALP. Finally, the crystal structure of ALP is required to validate our model and support our findings concerning Mn 2+ binding.
Considering that ALP has emerged as a central enzyme in regulating crucial neurological processes, a detailed understanding of its interaction with Mn 2+ and how its activity is controlled will be essential in defining this enzyme as a promising drug target to treat human afflictions [35,45].

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).

Enzyme Preparations
The sequence of central-ALP was amplified using the P fidelity DNA-polymerase) from the plasmid H6pQE60-29.2, co The desired sequence was confirmed by automated DNA sequ 630 bp (210 aa), was directionally cloned into the histidine-tag and the histidine-tagged proteins were expressed in E. coli str mM isopropyl-β-D-thiogalactopyranoside. The central-ALP wa cellulose anion exchange chromatography (calibrated with Tri 250 mM and an NTA-Ni 2+ affinity chromatography. The purity mutant D217A, the double mutant E288A/K290A, and the trip putative metal-ligand site of ALP were obtained by using the Q Kit (Stratagene) with the plasmid H6pQE60-29.2, containing th presence of the desired mutation and the absence of unwanted DNA sequence analysis. resolution), Clostridium difficile (PDB id: 3LHL, 22% identity, 32% similarity, 2.3 Agmatine, glycine, Tris, SDS, and all other reagents were of the highest qu available (most from Sigma Aldrich Chemical Co. Louis, MO, USA). Restriction e enzymes and reagents for PCR, were obtained from Invitrogen Co. (Carlsbad, CA, U nucleotide primers were obtained from the Fermelo Biotec Co. (Santiago, Chile).

Enzyme Preparations
The sequence of central-ALP was amplified using the PCR technique (with fidelity DNA-polymerase) from the plasmid H6pQE60-29.2, containing the ALP cDN The desired sequence was confirmed by automated DNA sequence analysis. The am 630 bp (210 aa), was directionally cloned into the histidine-tagged pQE60 bacteria and the histidine-tagged proteins were expressed in E. coli strain JM109, following mM isopropyl-β-D-thiogalactopyranoside. The central-ALP was partially purified b cellulose anion exchange chromatography (calibrated with Tris-HCl 10 mM, pH 7. 250 mM and an NTA-Ni 2+ affinity chromatography. The purity of all preparations w mutant D217A, the double mutant E288A/K290A, and the triple mutant, N213A/Q putative metal-ligand site of ALP were obtained by using the QuikChange ® Site-Di Kit (Stratagene) with the plasmid H6pQE60-29.2, containing the ∆LIM-ALP cDNA a presence of the desired mutation and the absence of unwanted changes were confir DNA sequence analysis. resolution), Burkholderia thailandensis (PDB id: 4DZ4, 22% identity, 31% similarity, 1.7

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

Enzyme Preparations
The sequence of central-ALP was amplified using the PCR technique (with Kod, Merck, fidelity DNA-polymerase) from the plasmid H6pQE60-29.2, containing the ALP cDNA as the temp The desired sequence was confirmed by automated DNA sequence analysis. The amplified fragme 630 bp (210 aa), was directionally cloned into the histidine-tagged pQE60 bacterial expression ve and the histidine-tagged proteins were expressed in E. coli strain JM109, following induction with mM isopropyl-β-D-thiogalactopyranoside. The central-ALP was partially purified by means of DE cellulose anion exchange chromatography (calibrated with Tris-HCl 10 mM, pH 7.5), eluted with 250 mM and an NTA-Ni 2+ affinity chromatography. The purity of all preparations was ~70%. The si mutant D217A, the double mutant E288A/K290A, and the triple mutant, N213A/Q215A/K290A o putative metal-ligand site of ALP were obtained by using the QuikChange ® Site-Directed Mutagen Kit (Stratagene) with the plasmid H6pQE60-29.2, containing the ∆LIM-ALP cDNA as the template. presence of the desired mutation and the absence of unwanted changes were confirmed by autom DNA sequence analysis.

Enzyme Preparations
The sequence of central-ALP was amplified using the PCR t fidelity DNA-polymerase) from the plasmid H6pQE60-29.2, containi The desired sequence was confirmed by automated DNA sequence a 630 bp (210 aa), was directionally cloned into the histidine-tagged p and the histidine-tagged proteins were expressed in E. coli strain JM mM isopropyl-β-D-thiogalactopyranoside. The central-ALP was par cellulose anion exchange chromatography (calibrated with Tris-HC 250 mM and an NTA-Ni 2+ affinity chromatography. The purity of all mutant D217A, the double mutant E288A/K290A, and the triple mu putative metal-ligand site of ALP were obtained by using the QuikC Kit (Stratagene) with the plasmid H6pQE60-29.2, containing the ∆LIM presence of the desired mutation and the absence of unwanted chan DNA sequence analysis. resolution), and a proclavaminate amidino hydrolase from Streptomyces clavuligerus (PDB id: 1GQ6, 22% identity, 33% similarity, 1,75

Enzyme Preparations
The sequence of central-ALP w fidelity DNA-polymerase) from the p The desired sequence was confirmed 630 bp (210 aa), was directionally clo and the histidine-tagged proteins we mM isopropyl-β-D-thiogalactopyran cellulose anion exchange chromatogr 250 mM and an NTA-Ni 2+ affinity chr mutant D217A, the double mutant E putative metal-ligand site of ALP we Kit (Stratagene) with the plasmid H6 presence of the desired mutation and DNA sequence analysis. resolution), several of them including Mn 2+ . The alignment of the templates was done by Clustal using BLOSUM45 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. The residues forming the site were proposed through structural alignment with the templates that included Mn 2+ in the structure.

Enzyme Preparations
The sequence of central-ALP was amplified using the PCR technique (with Kod, Merck, high fidelity DNA-polymerase) from the plasmid H6pQE60-29.2, containing the ALP cDNA as the template. The desired sequence was confirmed by automated DNA sequence analysis. The amplified fragment of 630 bp (210 aa), was directionally cloned into the histidine-tagged pQE60 bacterial expression vector, and the histidine-tagged proteins were expressed in E. coli strain JM109, following induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside. The central-ALP was partially purified by means of DEAE-cellulose anion exchange chromatography (calibrated with Tris-HCl 10 mM, pH 7.5), eluted with KCl 250 mM and an NTA-Ni 2+ affinity chromatography. The purity of all preparations was~70%. The single mutant D217A, the double mutant E288A/K290A, and the triple mutant, N213A/Q215A/K290A of the putative metal-ligand site of ALP were obtained by using the QuikChange ® Site-Directed Mutagenesis 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 DNA sequence analysis.

ALP Activity Determination
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 MnCl 2 . All the assays were initiated by adding the enzyme to the substrate, buffer, and MnCl 2 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.

Enzyme-Metal Interactions Analysis
For reactivation assays with Mn 2+ , the enzyme was incubated for 15 min at 37 • C with varying concentrations of Mn 2+ 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 constants (K a ) were estimated from the hyperbolic dependence of agmatinase activity on free-Mn 2+ concentrations, using nonlinear regression analysis in Graph Pad Prism 5.0 (similar to a K m for Mn 2+ ). Free Mn 2+ concentrations were calculated using a dissociation constant of 3.98 × 10 −8 M and 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].

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.