Guanidino acid hydrolysis by the human enzyme annotated as agmatinase

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.

. Sequence comparison and catalytic activity of human AGMAT variant R105. (A) Amino acid identity of the mature form of AGMAT compared to guandidinobutyrase (GbuA) and guanidinopropionase (GpuA) from Pseudomonas aeruginosa, agmatinase from Escherichia coli (SpeB) and human arginase 1 (ARG1). The numbers of identical (blue) and different (red) residues of the respective enzymes in comparison to AGMAT were derived from a multiple sequence alignment ( Supplementary Fig. S1). (B) Specific activity of recombinantly expressed and purified AGMAT in the presence of 10 mM GBA or agmatine (Agm). Column represents the average of technical triplicates and consistent results were obtained with independent preparations (Data from Fig. 2C). (n.d. = not detected; limit of detection ~ 0.02 nmol s -1 mg -1 ), error bars, s.d. www.nature.com/scientificreports/ or Arg1. Furthermore, we noted that guanidinobutyrase GbuA and guanidinopropionase GpuA from Pseudomonas aeruginosa 3,36 share considerably higher degrees of similarity with 60.2% and 45.2% identical amino acids, respectively with AGMAT. Since the activity of human AGMAT has never been demonstrated conclusively, we tested whether guanidinobutyrate is hydrolyzed more efficiently compared to the currently assumed substrate agmatine. In accordance with the activity of the more similar enzyme, we observed that recombinant AGMAT catalyzed the hydrolysis of GBA whereas agmatine hydrolysis was not detected (Fig. 1B).
The surprising discovery of guanidinobutyrate as substrate for the human agmatinase prompted us to test a variety of different substrates. We found that guanidino acids of a certain chain length such as guanidinobutyrate and taurocyamine are generally accepted as substrates for AGMAT ( Fig. 2A). In order to gain structural insights into the substrate preference of AGMAT, we built a model of the 3D-structure of a monomer using the Robetta server 37 . Members of the arginase or ureohydrolase family commonly form hexamers, where a neighboring subunit contributes to the formation of the substrate-binding groove in another subunit leading to a shared active site. To properly assess the selectivity of the enzyme and the features of the AGMAT active site, we modeled the local quaternary structure of the enzyme based on the arrangement of subunits in the crystal structure of GbuA (PDB entry 3NIO) 36 (Fig. 2B). For this, the catalytic residues of AGMAT and GbuA were superimposed to match two neighboring subunits as present in the biological assembly of GbuA. Ions, which are also not included in Robetta models, were placed according to their location relative to the active site residues in GbuA. The residues coordinating two Mn 2+ in the active site as well as His201 and Glu320 essential for catalytic activity in human arginase 38 are fully conserved between GbuA and AGMAT. In addition, eight out of 10 residues in close proximity to the substrate-binding pocket are conserved between AGMAT and GbuA ( Supplementary Fig. S1). Due to this high similarity, GbuA is an excellent template to evaluate the active site features of AGMAT. Within the GbuA structure, it can be seen that the active site of a subunit is capped by residue R72 from a neighboring subunit, so that this residue could interact with the carboxylate group of the substrate guanidinobutyrate. Interestingly, in the equivalent position to R72 in AGMAT (position 105) two variants exist, with either arginine or glycine in this position. In the genomes covered by the NCBI short genetic variation database, the G105 variant is slightly www.nature.com/scientificreports/ more abundant (57.7%) in the sampled human population compared to the R105 variant (42.3%) (dbSNP, entry rs6429757). Both variants have been used independently in studies aiming to demonstrate agmatinase activity of AGMAT 5,6 . To assess the possible influence of having a large, positively charged R105 residue instead of a small glycine, R105 was modeled using the rotamer observed in GbuA and taurocyamine, the best substrate in initial experiments, was docked into place in the active site using Autodock Vina. This complete substrate-bound AGMAT model shows that R105 can indeed reach into the active site of the neighboring subunit and potentially interacts with the sulfonic group of taurocyamine (Fig. 2B).
To compare the enzymatic activity and substrate specificity of both AGMAT variants (R105 and G105), we expressed the mature forms of the mitochondrial enzyme lacking the predicted N-terminal transit peptide of 35 amino acids (MitoFates 39 ) in E. coli and purified them by Ni-affinity chromatography ( Supplementary  Fig. S1). Consistent with previous studies, we observed no agmatine hydrolysis with both variants of purified AGMAT. In contrast, both variants efficiently hydrolized GBA ( Fig. 2A,C). When we provided a range of similar guanidine derivatives that differ in chain length and additional substitutions at a substrate concentration of 10 mM, both AGMAT variants additionally hydrolyzed TC, hypotaurocyamine (HTC, a reduced form of TC), and guanidinopropanesulfonate (GPS). For variant R105, the specific activity was three times higher for TC (1.4 nmol s −1 mg −1 ) compared to the second-best substrate GBA (0.4 nmol s −1 mg −1 ), whereas in comparison variant G105 showed reduced activity towards TC and slightly enhanced activity for GBA (Fig. 2C). As described above, when TC is placed into the structural model, its sulfonate group is in proximity of the guanidine group of R105 (Fig. 2B). Variant G105 hydrolyzed arginine to a small extent, whereas variant R105 was inactive with arginine but hydrolyzed GPA instead (Fig. 2C). Urea release from agmatine or other guanidine derivatives lacking a negatively charged group (mercaptoethylguanidine, methylguanidine, guanidine) was not detected, except for guanidinoethanol that was a poor substrate for variant G105. Similarly, guanidinosuccinate, guanidinoacetate and creatine were not hydrolyzed by either variant of AGMAT at a substrate concentration of 10 mM. Taken together, AGMAT exhibits a rather broad substrate specificity towards linear guanidino acids with intermediate chain lengths, but is almost strictly dependent on a negatively charged head group. Variant R105 prefers shorter substrates compared to variant G105 that even showed weak activity with arginine. In light of our results, we propose to re-annotate the human enzyme formerly known as agmatinase (AGMAT) as guanidino acid hydrolase (GDAH) to better reflect its enzymatic activity.
Of the identified substrates of GDAH, only TC, GBA and GPA have been detected in human samples according to the Human Metabolome Database (HMDB) 40 . For this reason, we determined the concentration dependence of GDAH activity for these three compounds. The Michaelis constants (K M ) for TC, GBA and GPA hydrolysis by GDAH were all > 50 mM for both, the GDAH variant R105 (Fig. 3A) and the variant G105 ( Supplementary  Fig. S1). The exact values could not be determined because no saturation was achieved within the concentration limits set by the solubility of the compounds. Throughout the concentration range tested, the specific activity for TC hydrolysis was approximately three times higher compared to GBA hydrolysis with variant R105, and GPA hydrolysis rates were substantially lower. GDAH was further characterized by using variant R105 with TC as best substrate. When GDAH was expressed in normal LB medium, its specific activity was low but the specific activity was increased four-fold when the protein was expressed in medium supplemented with 0.5 mM Mn 2+ (Fig. 3B), consistent with a dependence on Mn 2+ common to most members of the ureohydrolase family. The apparent pH optimum of GDAH was 10 and the apparent temperature optimum 67 °C (Fig. 3C,D). We determined the activation energy for TC hydrolysis to be 46 ± 1 kJ/mol (Fig. 3D).
GDAH substrates are not used as alternative phosphagens. Various guanidine derivatives ranging from arginine to creatine are used as phosphagens in different organisms, serving as cellular buffers of high energy phosphates. Phosphorylation of the guanidine group of TC yields phosphotaurocyamine, which is used as phosphagen in marine annelids and some protists 41,42 . In order to shed light on potential physiological roles of the identified GDAH substrates TC, GBA and GPA we tested whether these guanidine derivatives could serve as so far unrecognized phosphagens in humans by getting phosphorylated by one of the human creatine kinase isoforms. For this purpose, we overexpressed and purified the four charaterized human creatine kinase isoenzymes 43,44 . The creatine kinases were incubated with γ-32 P-ATP as phosphate donor and creatine, TC, GBA, or GPA as substrates. Analysis of the reaction products by thin-layer chromatography revealed that all four creatine kinases accepted exclusively creatine as substrate (Fig. 4A). As a control we overexpressed and purified the TC kinase from Arenicola brasiliensis 41 . With this enzyme we observed a phosphorylation product specifically with TC as substrate under the same assay conditions utilized for the creatine kinases ( Supplementary Fig. S1). These results indicate that the physiological function of GDAH is not concerned with the modulation of the levels of potential alternative phosphagens.
GATM synthesizes GDAH substrates. GBA, GPA and TC have been detected in human fluids but an endogenous source has so far not been identified. However, all three compounds have been shown previously to be generated in side reactions of mitochondrial glycine amidinotransferase in rats from the respective amino homologs γ-aminobutyric acid (GABA), β-alanine and taurine 45 . In order to clarify whether the identified substrates for GDAH could be produced by an endogenous activity, we overexpressed and purified human mitochondrial glycine amidino transferase (GATM) lacking the mitochondrial transit peptide (uniprot entry: P50440) and performed activity assays with the potential substrates. Similar to the previously characterized enzyme from rat, human GATM had the highest activity with glycine, but also GABA, β-alanine and taurine were accepted as substrates with decreasing preference (Fig. 4B) www.nature.com/scientificreports/ Discussion 20 years ago, a human gene from the ureohydrolase family has been annotated as agmatinase. However, we demonstrate that GDAH/AGMAT hydrolyzes guanidino acids rather than agmatine (Fig. 2C). Human GDAH has a high degree of sequence similarity with the guanidinobutyrase GbuA of Pseudomonas aeroguinosa and its three-dimensional structure can be predicted with high confidence based on the GbuA structure (PDB 3NIO).  www.nature.com/scientificreports/ Two enzyme variants exist in humans in regard to residue 105 that likely participates in the binding of the substrates. In 57.7% of human sequences arginine is replaced by glycine at this position. Our modeling based on the P. aeroguinosa guanidinobutyrase and subsequent docking of TC suggests that R105 can extend into a neighboring subunit's active site, positioning R105 to directly contact the negatively charged head group of the substrate. Although we observed residual arginine hydrolysis by GDAH variant G105, agmatine was not hydrolyzed by both tested GDAH variants. Instead, both GDAH variants hydrolyzed TC and GBA efficiently. Variant R105 exhibited the highest activity with TC, whereas variant G105 turned over GBA and TC to the same extent. Both GDAH variants also hydrolyzed GPA but for the G105 variant, this activity was only detectable at very high substrate concentrations far above the concentrations detected in human samples that are in the low µM range (HMDB) 40 . Still, we cannot exclude that concentrations might be higher in cells or certain compartments such as the mitochondria, where both GDAH and GATM are predicted to be localized (NCBI Gene database). Overall, variant G105 preferred longer substrates compared to variant R105 and even accepted arginine and guanidinoethanol as poor substrates. Both K M and reaction rate for all substrates are representative of only low to intermediate enzymatic activity. However, kinetic constants in this range do not allow to draw conclusions about a potential physiological relevance. For example, we recently reported that despite its relatively high K M the guanidine hydrolase GdmH allowed Synechocystis to utilize guanidine as sole nitrogen source 8 . The analysis of further substrates that are not known to occur in humans indicated that the activity of GDAH strictly depends on a negatively charged functionality opposite to the guanidine group. The first report of an enzymatic activity of this enzyme allowed a more thorough characterization: GDAH shares common features such as rather high K M values, Mn 2+ -dependency, and high apparent temperature and pH optima with other members of the ureohydrolase superfamily 46 . We show that GDAH substrates GBA, GPA and TC were not accepted as substrates of human creatine kinases but could be formed by the action of human GATM. GATM promiscuously transferred the amidino group of arginine to glycine, GABA, β-alanine and taurine yielding the respective guanidine compounds GBA, GPA and TC as it has been demonstrated for some mammalian homologs before 45,47 . Expression data and subcellular localization suggest that GDAH and GATM co-localize in the mitochondria of kidney, liver and brain. Even if taurine is a minor substrate of GATM, TC could be formed in substantial amounts via this reaction as taurine is produced in the liver and reaches concentrations of 10-50 mM 48 . In addition to the activity of GATM, the identified guanidino acids could be produced by so-far unknown metabolic processes. GBA could be an intermediate of agmatine/arginine catabolism via guanidinobutyraldehyde 49 . However, the activity of diamine oxidase towards agmatine to produce guanidinobutyraldehyde is controversial 50 . Alternatively, GBA, GPA and TC could originate from direct uptake from the diet 32,33 or from gut microbial activities 34 .
TC has been used since the late 1970's to manipulate taurine levels in different organs or tissues since it is a competitive inhibitor of the taurine transporter TAUT 51,52 . Additionally, TC has been reported as an antagonist of GABA A and glycine receptors [28][29][30][31] . GBA has been detected in the brain, liver and kidney of vertebrates and insects and elevated levels of GBA and TC in the brain of rabbits have been related to convulsions 25,26 . Given these effects of the identified GDAH substrates, a possible function of GDAH could be the hydrolysis of potentially deleterious concentrations of guanidino acids as a means of detoxification. This hypothesis would also agree with the observed broad substrate specificity of GDAH. GDAH expression has been reported to be reduced in diabetic patients with breast cancer or clear cell type of renal cell carcinoma 53,54 . GDAH variant R105 of GDAH has been linked to type 1 diabetes 55 . On the other hand, GDAH was reported to promote lung adenocarcinoma tumorigenesis and the progression of colorectal cancer by inducing chronic inflammation 56,57 .
The potential involvement of GDAH/AGMAT in agmatine metabolism has received increasing attention from neuroscientists starting in 1994 when agmatine had been detected in the human brain 16 . Agmatine is discussed to have beneficial effects on disorders of the central nervous system like depression, nerve regeneration and epilepsy among others and is generally believed to be neuroprotective 7,17 . GDAH is highly expressed in kidney, liver and hippocampal interneurons [58][59][60] . The presented findings implicate that GDAH is unlikely to contribute directly to the modulation of endogenous agmatine concentrations. Further evidence for a physiological role of GDAH as guanidino acid hydrolase comes from two recent observations: Concentrations of GBA and GPA have been found to negatively correlate with the expression level of GDAH in a genome-wide study of genes affecting metabolite concentrations in saliva 15 . In addition, a further study identified an association of altered concentrations of GPA with genetic variations in the chromosomal locus of GDAH 61 . In conclusion, the findings presented in this study revise some fundamental aspects of the metabolism of guanidine derivatives in humans and pose further questions regarding the functional or pathological roles of GDAH and its substrates in human physiology.

Methods
Bacterial cultivation, cloning, protein overexpression and purification. E. coli BL21(λDE3) gold (Invitrogen) or E. coli SoluBL21 (Genlantis) were grown in LB with 50 µg/ml kanamycin when transformed. Genes of interest (human GDAH, GATM, CKM, CKB, CKMT1, and CKMT2, as well as TCK from Arenicola brasiliensis 41 ) were codon optimized for E. coli and synthesized by GeneArt (LifeTechnologies). Mitochondrial target sequences were predicted with MitoFates 39 and excluded to obtain mature enzymes. DNA constructs were directly cloned into a pET24 derivative by Gibson assembly to obtain enzymes with an N-terminal 6xHis-tag followed by a TEV cleavage site. For recombinant protein expression, E. coli BL21(λDE3) gold (Invitrogen) or E. coli SoluBL21 (Genlantis), in case of GDAH and CKMT1, were transformed with the expression constructs, grown in LB supplemented with 0.5 mM MnCl 2 , if not stated otherwise, at 37 °C to an OD 600 of 0.6, transferred to 18 °C and induced over night with 1 mM IPTG. The cells were harvested by centrifugation, resuspended in enzyme buffer (50 mM Tris-HCl pH 8, 100 mM NaCl) supplemented with 1 × EDTA-free cOmplete protease inhibitor (Roche) and lysed by ultrasonication (Branson). After centrifugation at 12,000 g for 20 min at 4 °C, the soluble www.nature.com/scientificreports/ protein fraction was incubated with His-bind NiNTA resin (Qiagen, Hilden, Germany) for 30 min at 4 °C. The resin was loaded into gravity flow columns and washed sequentially with enzyme buffer supplemented with 20 mM or 50 mM imidazole. The His-tagged enzymes were eluted in enzyme buffer supplemented with 500 mM imidazole and desalted into enzyme buffer by passage through PD50 columns (GE lifesciences).
Modeling. A comparative model of AGMAT was produced using the Robetta server 37 . This produced a model containing just one subunit. Therefore, to reconstitute the oligomeric state, the AGMAT model was aligned to chain A of 3NIO using the catalytic site residues (H129, D152, H154, D156 and D243 of 3NIO). A second copy of the AGMAT model was then structurally aligned to chain B of 3NIO. The active site manganese ions were modeled by using the positions of the ions in 3NIO relative to the active site residues. In the produced model R105 was modeled to have the rotameric conformation of R72 in PaGbuA. The AGMAT models were prepared for docking by removing the hydrogens from original Robetta models and re-adding and reparametrizing them using Chimera UCSF Dock Prep. Hydrogens and charges were calculated using ANTECHAMBER 62 . The AMBER ff14SB forcefield 63 was applied for standard residues and Gasteiger for other residues 64  Glycine amidinotransferase assay. 3.4 µg glycine amidinotransferase was incubated in a buffer containing 80 mM KCl, 20 mM NaCl, 2 mM MgCl 2 , 80 mM Tris (pH 8) with 10 mM arginine and 10 mM of the indicated substrate. The ornithine concentration was measured as described 67,68 . In brief, 100 µl sample were mixed with equal volumes of both ninhydrin solution (25 mg ml -1 ninhydrin in 60% (v/v) acetic acid and 13.8% (w/v) phosphoric acid) and glacial acetic acid. The reaction mixture was incubated for 15 min at 96 °C. Reactions were chilled and the absorption at 520 nm was measured in a 96-well plate (TECAN Spark Reader).
Non-commercial guanidine compounds. Synthesis and characterization of commercially unavailable guanidino compounds is described in the supplementary information.

Data availability
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