biology

Review Ornithine Aminotransferase, an Important Glutamate-Metabolizing at the Crossroads of Multiple Metabolic Pathways

Antonin Ginguay 1,2, Luc Cynober 1,2,*, Emmanuel Curis 3,4,5,6 and Ioannis Nicolis 3,7

1 Clinical Chemistry, Cochin Hospital, GH HUPC, AP-HP, 75014 Paris, France; [email protected] 2 Laboratory of Biological Nutrition, EA 4466 PRETRAM, Faculté de Pharmacie, Université Paris Descartes, 75006 Paris, France 3 Laboratoire de biomathématiques, plateau iB2, Faculté de Pharmacie, Université Paris Descartes, 75006 Paris, France; [email protected] (E.C.); [email protected] (I.N.) 4 UMR 1144, INSERM, Université Paris Descartes, 75006 Paris, France 5 UMR 1144, Université Paris Descartes, 75006 Paris, France 6 Service de biostatistiques et d’informatique médicales, hôpital Saint-Louis, Assistance publique-hôpitaux de Paris, 75010 Paris, France 7 EA 4064 “Épidémiologie environnementale: Impact sanitaire des pollutions”, Faculté de Pharmacie, Université Paris Descartes, 75006 Paris, France * Correspondence: [email protected]; Tel.: +33-158-411-599

Academic Editors: Arthur J.L. Cooper and Thomas M. Jeitner Received: 26 October 2016; Accepted: 24 February 2017; Published: 6 March 2017

Abstract: Ornithine δ-aminotransferase (OAT, E.C. 2.6.1.13) catalyzes the transfer of the δ-amino group from ornithine (Orn) to α-ketoglutarate (aKG), yielding glutamate-5-semialdehyde and glutamate (Glu), and vice versa. In mammals, OAT is a mitochondrial enzyme, mainly located in the liver, intestine, brain, and kidney. In general, OAT serves to form glutamate from ornithine, with the notable exception of the intestine, where citrulline (Cit) or arginine (Arg) are end products. Its main function is to control the production of signaling molecules and mediators, such as Glu itself, Cit, GABA, and aliphatic polyamines. It is also involved in proline (Pro) synthesis. Deficiency in OAT causes gyrate atrophy, a rare but serious inherited disease, a further measure of the importance of this enzyme.

Keywords: ornithine aminotransferase; glutamate; ornithine; gyrate atrophy

1. Introduction Ornithine δ-aminotransferase (E.C. 2.6.1.13; OAT), or ornithine δ-, is an enzyme found in almost all eukaryotic organisms, from protozoans to humans, and from fungi to higher plants. It is strongly conserved among these organisms. In humans, OAT deficiency causes gyrate atrophy, a serious inherited disease. This suggests that OAT plays an important role in metabolism. In most situations, it uses L-ornithine (Orn) as a , with L-glutamate (Glu) as an end ; OAT therefore belongs to what is termed the “glutamate crossway” [1]. At first sight, the importance of OAT may appear to be related to the roles of Glu in metabolism (bioenergetics, nitrogen flux, acid-base homeostasis), like the major involved in Glu metabolism (e.g., aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and glutamate dehydrogenase (GDH)). This paper summarizes current knowledge on OAT, in particular in the light of recent advances in large scale analysis, and of clinical experience, and argues that viewing the role of OAT as limited to glutamate-related energy metabolism, especially in mammals, probably misses other important functions of OAT. This is evident from an examination of the pathways relating glutamate and ornithine

Biology 2017, 6, 18; doi:10.3390/biology6010018 www.mdpi.com/journal/biology Biology 2017, 6, 1 2 of 40

BiologyornithineBiology 20172017 , 6,to,6 1, 18several key molecules in cell metabolism and “fate” regulation (Figure 1); 2these 2of of 40 38 functions are briefly summarized here—a detailed description lies outside the scope of this paper, ornithinebut interested to several readers key can molecules find information in cell metabolism in recent andreviews “fate” [2 –regulation5]. To present (Figure the 1)state; these of to several key molecules in cell metabolism and “fate” regulation (Figure1); these functions are briefly functionsknowledge are on briefly OAT, summarizedwe will first describe here—a thedetail physiced descriptional, chemical lies and outside biological the characteristicsscope of this paper, of the summarized here—a detailed description lies outside the scope of this paper, but interested readers butOAT interested reaction, andreaders then gocan on find to consider information the involvement in recent reviewsof OAT in[2 pathological–5]. To present states the or diseases.state of can find information in recent reviews [2–5]. To present the state of knowledge on OAT, we will first knowledgeThis systematic on OAT overview, we will will first alsodescribe highlight the physic needals, chemicalfor further and studies biological of thischaracteristics enzyme and of theits describe the physical, chemical and biological characteristics of the OAT reaction, and then go on to OATregulation. reaction , and then go on to consider the involvement of OAT in pathological states or diseases. Thisconsider systematic the involvement overview ofwill OAT also in pathologicalhighlight need statess for or diseases.further studies This systematic of this overviewenzyme and will alsoits regulation.highlight needs for further studies of this enzyme and its regulation.

Figure 1. End products of the reaction mediated by ornithine (Orn) aminotransferase (OAT) and theirFigure functions 1. End (in products italics) ofGABA: the reaction γ-aminobutyric mediated acid, byornithine Glu: glutamate, (Orn)aminotransferase Pro: proline, Arg: (OAT) arginine, and FigureCit:their citrulline, functions1. End Put:products (in putrescine, italics) of GABA:the NO: reaction γnitric-aminobutyric mediated oxide. by acid, ornithine Glu: glutamate, (Orn) aminotransferase Pro: proline, Arg: (OAT) arginine, and theirCit: functions citrulline, Put:(in italic putrescine,s) GABA: NO: γ-aminobutyric nitric oxide. acid, Glu: glutamate, Pro: proline, Arg: arginine, 1.1. PhysicCit: citrulline,al And Chemical Put: putrescine, Aspects NO: of the nitric Oat oxide-Mediated. Reaction 1.1. Physical And Chemical Aspects of the Oat-Mediated Reaction 1.11.1.1. Physic. Reactional And Chemical Aspects of the Oat-Mediated Reaction 1.1.1. Reaction 1.1.1. OATReaction catalyzes the conversion of Orn into glutamyl-5-semi-aldehyde (GSA) and vice versa, using OATaKG and catalyzes Glu as the cosubstrates conversion (Figure of Orn 2). into This glutamyl-5-semi-aldehyde reaction has an apparent equilibrium (GSA) and constant, vice versa, K [Glu][GSA] =using OAT aKG ,catalyzes that and favor Glu the ass the cosubstratesconversion degradation of (Figure ofOrn Orn: into2). experimentally, This glutamyl reaction-5-semi has K lies an-aldehydeapparent between (GSA) equilibrium50 and and 70 viceat constant,25 versa°C [6], using[Orn[ ]Glua[aKGKG][GSA] and] Glu as cosubstrates (Figure 2). This reaction has an apparent equilibrium constant,◦ K K = [Orn][aKG] , that favors the degradation of Orn: experimentally, K lies between 50 and 70 at 25 C[6] and[Glu pH][GSA 7.] 1. Even so, when Orn is at a very low concentration, or GSA in large excess, the reaction =and pH 7.1., that Even favor so,s the when degradation Orn is ata of very Orn: low experimentally, concentration, K orlies GSA between in large 50 and excess, 70 at the 25 reaction °C [6] can[Orn proceed][aKG] towards Orn synthesis, which has indeed been demonstrated in vitro by Strecker et al. can proceed towards Orn synthesis, which has indeed been demonstrated in vitro by Strecker et al. [6]. and[6]. OATpH 7. can1. Even thus so,catalyze when the Orn reaction is at a invery either low direction, concentration, dependi or ngGSA on inthe large relative excess, amounts the reaction of each OAT can thus catalyze the reaction in either direction, depending on the relative amounts of each cansubstrate proceed present, towards and Orn their synthesis use by ,subsequent which has indeedenzymes. been This demonstrated observation isin thevitro first by indication Strecker et that al. substrate present, and their use by subsequent enzymes. This observation is the first indication that [6]OAT. OAT stands can atthus the catalyze crossroad thes ofreaction several in metabolic either direction, pathways. dependi ng on the relative amounts of each substrateOAT stands present, at the and crossroads their use of by several subsequent metabolic enzymes. pathways. This observation is the first indication that OAT stands at the crossroads of several metabolic pathways.

Figure 2. Overview of the reaction catalyzed by OAT. Figure 2. Overview of the reaction catalyzed by OAT. 1.1.2. A Key Feature: Spontaneous Cyclization of GSA 1.1.2. A Key Feature: SpontaneousFigure 2. Overview Cyclization of the of GSAreaction catalyzed by OAT. Since GSA contains both an aldehyde and an amine function, internal imination can occur (Figure3), leading to a chemical equilibrium with the cyclic form ( S)-∆1-pyrroline-5-carboxylate 1.1.2. A Key Feature: Spontaneous Cyclization of GSA

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Since GSA contains both an aldehyde and an amine function, internal imination can occur (Figure 3), leading to a chemical equilibrium with the cyclic form (S)-Δ1-pyrroline-5-carboxylate (P5C). In addition, the aldehyde can react with water to give a GSA hydrated form, as shown in Figure 3. It has been suggested since the 1930s that the cyclic forms of these types of compounds are Biologyfavored2017 for, 6, 18entropic reasons, as is often the case for similar reactions (almost all oses), at least3 of 38at physiological pH [7,8], but the exact equilibrium point is difficult to establish because P5C undergoes polymerization [9]. For a similar compound, Δ1-pyrroline-2-carboxylate (P2C), a (P5C).dependence In addition, on pH thehas aldehydebeen demonstrated, can react withwith waterthe cyclic to giveform a being GSA the hydrated only detec form,table as one shown at pH in Figureabove 37. [10] It has. Despite been suggestedproblems with since stability the 1930s the thatdependence the cyclic of formsthe opening of these of typesthe cyclic of compounds form of P5C areon pH favored has been for entropic accomplished reasons, by as nuclear is often magnetic the case forresonan similarce ( reactionsNMR) [11] (almost, using all deuterated oses), at least water. at physiologicalThe two equilibria pH [7, 8depend], but the on exact pH: equilibrium At pD below point 6.2 is difficultthe free toaldehyde establish GSA because and P5C the undergoes hydrated 1 polymerizationgem-diol linear forms [9]. For predominate a similar compound,, the free aldehyde∆ -pyrroline-2-carboxylate increasing over the hydrated (P2C), a dependencegem-diol as the on pHpH hasis lowered been demonstrated,. The free aldehyd withe becomes the cyclic negligible form being at pD the above only detectable 4.9. At pD one6.2 a at sharp pH above transition 7 [10 is]. Despitedetected problems in the NMR with signal, stability andthe the dependencecyclic P5C imine of the form opening becomes of the predominant cyclic form above of P5C neutral on pH pH. has beenThus, accomplished in the by nuclear matrix, magnetic where resonance the pH (NMR) is around [11], using 7.8, the deuterated GSA produced water. The by twothe equilibriaOAT-mediated depend ornithine on pH: Atcatabolism pD below cyclizes 6.2 the freespontaneously aldehyde GSA towards and the P5C, hydrated making gem-diol the reaction linear formsalmost predominate, irreversible in the vitro free [12] aldehyde. The computed increasing [13] over pK thefor the hydrated amine gem-diolgroup protonation as the pH is is 9; lowered. thus at The free aldehyde becomes negligible at pD above 4.9. At pD 6.2 a sharp transition is detected in pH below 7.2, the -NH2 group is protonated and is not available for the imination cyclization. theHowever, NMR signal,since the and reaction the cyclic reaches P5C equilibrium imine form becomesquickly, even predominant a minimal above amount neutral of linear pH. GSA Thus, can in thebe used mitochondrion by OAT for matrix, the reverse where direction, the pH and is around drive the 7.8, decycli the GSAzation produced of P5C byinto the GSA. OAT-mediated In addition, ornithinethe mitochondrion catabolism matrix cyclizes is a spontaneously far more complex towards medium P5C, than making an aqueous the reaction solution, almost with irreversible a rather inlow vitro water[12 ].content The computed; hence the [13 ]equilibrium pK for the aminebetween group GSA, protonation hydrated isGSA 9; thus and at P5C pH belowmay be 7.2, quite the -NHdifferent2 group from is protonated that observed and in is water. not available for the imination cyclization. However, since the reaction reachesThis equilibrium equilibrium quickly, between even GSA a minimal and amountP5C also of places linear GSAOAT can at bea usedmetaboli by OATc crossroad for the reverses, P5C direction,opening the and way drive to the proline decyclization (Pro) and of P5Crelated into metabolic GSA. Inaddition, pathways, the while mitochondrion GSA can matrixbe further is a farconverted more complex into glutamate medium and than into an related aqueous metabolites. solution, with a rather low water content; hence the equilibrium between GSA, hydrated GSA and P5C may be quite different from that observed in water.

Figure 3. Cyclization and hydration of glutamate-5-semi-aldehyde (GSA). P5C: pyrroline-5-carboxylate. Figure 3. Cyclization and hydration of glutamate-5-semi-aldehyde (GSA). P5C: pyrroline-5-carboxylate. This equilibrium between GSA and P5C also places OAT at a metabolic crossroads, P5C opening the1.1.3. way Kinetic to proline Characteristics (Pro) and of related OAT metabolic pathways, while GSA can be further converted into glutamate and into related metabolites. The basic kinetic properties of OAT have been extensively studied. Some properties are very 1.1.3.well conserved Kinetic Characteristics among species, of OAT as seen in Table 1, which summarizes the known kinetic constants. Of note, available values concern almost exclusively the reaction in the direction of ornithine The basic kinetic properties of OAT have been extensively studied. Some properties are very degradation; only one publication gives Km values for GSA (1.4 mM) and Glu (25 mM) for the rat well conserved among species, as seen in Table1, which summarizes the known kinetic constants. liver enzyme [14]. However, in mammals OAT exhibits very distinct properties depending on the Of note, available values concern almost exclusively the reaction in the direction of ornithine tissue it is isolated from, with major differences between the intestine, kidney and liver. Differences degradation; only one publication gives K values for GSA (1.4 mM) and Glu (25 mM) for the rat liver are marked enough to have elicited longm debate on whether isozymes exist, but since there is now enzyme [14]. However, in mammals OAT exhibits very distinct properties depending on the tissue it is agreement that there is only one for OAT, pseudo- not being transcribed or leading to isolated from, with major differences between the intestine, kidney and liver. Differences are marked non-functional proteins (see below), other explanations have to be found for these apparent enough to have elicited long debate on whether isozymes exist, but since there is now agreement discrepancies. that there is only one gene for OAT, pseudo-genes not being transcribed or leading to non-functional proteins (see below), other explanations have to be found for these apparent discrepancies.

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Table 1. Summary of the kinetic properties of OAT according to the tissue and the organism. Values of the Michaelis constant, Km, are given in mM.

Michaelis constant (Km) Activity Organ Tissue Species Optimal pH Reference Orn aKG Value Unit nmol P5C/mg Eye Retina Human 7.6–8.0 3.7 1.72 218 ± 22 [15] protein/h Bovine 5.6 1.3 [16] nmol P5C/mg Calf 234 ± 26 [15] protein/h nmol P5C/mg Rat 324 ± 43 [15] protein/h nmol P5C/mg Rabbit 240 ± 24 [15] protein/h Iris (ciliary nmol P5C/mg Rat 308 ± 26 [15] body) protein/h nmol P5C/mg Rabbit 227 ± 28 [15] protein/h nmol P5C/mg Calf 218 ± 26 [15] protein/h nmol P5C/mg Human 165 ± 17 [15] protein/h Liver Rat 2.8 [6] nmol P5C/mg 458 ± 56 [15] protein/h µmol/mg 7.8 2.7 3.0 11 [17] protein/min *** 8.0 0.56 0.91 [18] 8.15 [19] 1 0.7 [14] µmol/mg Mouse 7.5 4,8 0.7 10,8 [17] protein/min µmol P5C/mg ** 1.2 ± 0.11 2 ± 0.5 0,75 [20] proteine/h nmol P5C/mg Rabbit 317 ± 34 [15] protein/h Trout ** 7.3 7.5 ± 0.8 0.88 ± 0.2 0,033 µmol/min [21] Isolated Rat * 4 0,15 25 nmol/min/mg mitochondria protein [22] Kidney Rat *** 8.0 0.59 0.91 920 units/mg protein [18] nmol P5C/mg Rabbit 674 ± 48 [15] protein/h nmol P5C/mg Brain Rat 488 ± 42 [15] protein/h Rat 8 1,67 0,5 [23] nmol P5C/mg Rabbit 112 ± 18 [15] protein/h nmol P5C/mg 69 ± 18 [15] protein/h µmol P5C/mg Mouse ** 1.1 ± 0.1 2.6 1.1 [20] protein/h Cortical Mouse * 0.8 ± 0.3 [24] interneurons Cerebellar Mouse * 4.7 ± 0.9 [24] granule cells Astrocytes Mouse * 4.3 ± 2.2 [24] Small Rat *** 8.0 0.60 0.95 [18] intestine

Modiolus demissus [25] Plasmodium ± ± falciparum 3.95 1.04 0.65 0.21 [26] Pea (Pisum 8.8 15 2 60.4 nmol/mg/s [27] sativorum) Mothbean (Vignia 8 2 0.75 [28] aconitifolia) * unpurified enzyme, ** partial purification, *** crystallized enzyme.

Several possible explanations will be detailed in what follows, including alternative splicing [29], changes in OAT concentrations leading to different aggregation states with different properties [30], and changes in and succinylation states. OAT requires pyridoxal 5-phosphate (PLP) to be functional. OAT, like every PLP-dependent transaminase, operates via a “ping-pong” mechanism, two half-reactions completing a full transamination cycle. [12], as shown in Figure4. Like other , OAT in the absence of substrates forms an internal aldimine with the PLP covalently bound on a lysine residue through a Schiff base. In the first half-reaction, ornithine forms an external aldimine with PLP, no longer Biology 2017, 6, 1 5 of 40

Plasmodium 3.95 ± 0.65 ± [26] falciparum 1.04 0.21 Pea (Pisum 8.8 15 2 60.4 nmol/mg/s [27] sativorum) Mothbean (Vignia 8 2 0.75 [28] aconitifolia) * unpurified enzyme, ** partial purification, *** crystallized enzyme. Several possible explanations will be detailed in what follows, including alternative splicing [29], changes in OAT concentrations leading to different aggregation states with different properties [30], and changes in acetylation and succinylation states. OAT requires pyridoxal 5-phosphate (PLP) to be functional. OAT, like every PLP-dependent transaminase, operates via a “ping-pong” mechanism, two half-reactions completing a full transaBiology 2017mination, 6, 18 cycle. [12], as shown in Figure 4. Like other transaminases, OAT in the absence5 of of 38 substrates forms an internal aldimine with the PLP cofactor covalently bound on a lysine residue through a Schiff base. In the first half-reaction, ornithine forms an external aldimine with PLP, no longercovalently covalently bound tobound the enzyme, to the butenzyme retained, but inretained the active in sitethe throughactive site non-covalent through non interactions.-covalent δ Theinteractions. Orn -amino The Orn group δ-amino transfer group then tran transformssfer then transforms PLP into PLP a pyridoxamine into a pyridox phosphateamine phosphate (PMP) (PMP)intermediate, intermediate, thus forming thus forming a glutamate a glutamate semialdehyde-PMP semialdehyde aldimine.-PMP Thisaldimine. intermediate This intermediate is hydrolyzed is hydrolyzedto release GSA, to release leaving GSA, the OAT leaving complexed the OAT with complexed PMP. In with the second PMP. In half-reaction, the second aKGhalf-reaction forms a, Schiff aKG formsbase with a Schiff the PMP. base The with amino the PMP. group The is transferred amino group to aKG is transferred to form Glu, to while aKG theto cofactorform Glu, reverts while to the its ω α cofactorPLP form reverts [31]. OAT to its thus PLP acts form as an[31].-transaminase OAT thus acts in as the an first ω-transaminase half-reaction, andin the as anfirst -transaminasehalf-reaction, α andin the as second an α-transaminase half-reaction: in although the second the half-amino-reaction group: although is more the reactive α-amino then group the distalis more one, reactive in the thenfirst half-reactionthe distal one, OAT in transaminatesthe first half-reaction the distal OAT OAT transaminates amino group the [32 ].distal The structuralOAT amino reason group for [32] this. ω The-transamination structural reason selectivity for this willω-transamination be explored below selectivity in 2.3. will be explored below in 2.3.

Figure 4 4.. “Ping“Ping-pong”-pong” mechanism mechanism half half-reactions-reactions of OAT of OAT (E-PLP: (E-PLP: OAT OAT PLP PLPadduct; adduct; E-PMP: E-PMP: OAT PMPOAT PMPadduct) adduct).

Each half-reaction has its own kinetic profile, with different effects of pH: for the first one, the Each half-reaction has its own kinetic profile, with different effects of pH: for the first one, reaction rate is maximal in the range pH 8–10; for the second one, it peaks at pH 6–8.5 [19]. The enzyme’s the reaction rate is maximal in the range pH 8–10; for the second one, it peaks at pH 6–8.5 [19]. optimal pH is thus a compromise: it depends on the organism considered (Table 1), owing to slight The enzyme’s optimal pH is thus a compromise: it depends on the organism considered (Table1), variations in the amino acid content of the protein, but is around 8, not far from the mitochondrial pH. owing to slight variations in the amino acid content of the protein, but is around 8, not far from the mitochondrial pH. 1.2. Inhibitors 1.2. InhibitorsBesides helping us to understand the mechanisms of action of OAT and its involvement in Biology 2017, 6, 1 6 of 40 metabolism,Besides recent helping advances us to understandon the role of the OAT mechanisms in cancer and of actionin other of pathological OAT and its process involvementes (see last in partmetabolism, of this paper) recent have advances fueled interest on the in role OAT of inhibitors OAT inConditions cancer [33,34]. and OAT in is other inhibited pathological by aminohexanoate processes α-Amino(see[35], lastGABAacids part, gabaculine of this paper), 5-fluoromethylornithine have fueled interest (5-FMOrn) in OAT [20,36 inhibitors–38], and [33 ,34L-canaline]. OAT [39] is . inhibited A more complete list of inhibitors and their structures is given in Table8 U OAT, 2. 37 by aminohexanoate [35], GABA, gabaculine, 5-fluoromethylornithine°C, pH 8, (5-FMOrn) [20,36–38], and Biology 2017, 6, 1 6 of 40 L-canaline [39]. A more complete list of inhibitors and their0.05 structuresmM is given in Table2. Table 2. OAT inhibitors described for mammalian enzyme. PO43− buffer Rat L-Canaline Irreversible Conditions Ki: 2 µM [39] 0.1 mM PLP, (liver) Table 2. OAT inhibitors described for mammalianExperiment enzyme.Ki or % OAT Referen α-Amino acidsInhibitor Structure Nature 175 mM 8 U OAT, 37 al Inhibition Origin ce Orn, 35 mM K or % OAT Inhibitor Structure Nature Experimental i Reference °C,Conditions pH 8, Inhibition Origin aKG α-Amino acids pH0.05 8, mM 0.05 8 U3− OAT, 37 ◦C, PO4 buffer3− Rat L-Canaline Irreversible mMpH 8,PO 0.054 mM Ki: 2 µM [39] Irreversible, 0.1 mM3 −PLP, (liver) Rat L-Canaline Irreversible bufferPO4 0.01buffer K : 2 µM Rat [39] noncompetit 1750.1 mM mM PLP, i (liver) [40] mM175 PLP, mM 20 Orn, (liver) ive Orn,mM35 Orn,35 mM mM aKG10 pHaKG 8, 0.05 mM Irreversible, 3− Rat POmM4 aKGbuffer 0.01 [40] noncompetitive pHmM 8, PLP, 0.05 20 mM (liver) 37Orn, °C, 10 pH mM aKG mM ◦PO43− Irreversible, 7.1,37 50C, mM pH 7.1, 3− buffer503− mM 0.01 PO 4 Rat Rat L -Canavanine noncompetit PO4 buffer 100%100% at 25 at 25 mMRat [40] [6] L-Canavanine mMbuffer PLP, 35 20 mM (liver) (liver) [6] ive 35 Orn,mM 3.75Orn, mM mM (liver) aKG,K mM3.75 Orn, mM 10 mM0.5 aKG U OAT; Rat aKG,K 23% at 20 mM [41] 50 mM Orn (kidney) 37 °C, pH Rat 0.5 U OAT; 23% at 20 7.1, 50 mM (kidne [41] PO50 4mM3− buffer Orn 100%mM at 25 Rat L-Canavanine y) [6] 3537 mM °C, Orn,pH mM (liver) 7.1,3.75 50 mM mM aKG,K PO43− buffer 59% at 50 Rat L-Norvaline Rat [6] 350.5 mM U OAT; Orn, 23%mM at 20 (liver) (kidne [41] 503.75 mM mM Orn mM aKG,K y) pH37 8,°C, 50 pH mM 7.1, 50 mM PO43− buffer PO43− buffer 59%50% atat 5010 Rat L-Norvaline 0.1 mM PLP, [14][6] mM (liver) 3510 mMmM Orn,Orn, mM (liver) 53.75 mM mM aKG aKG,K Rat 0.5 U OAT; 44% at 100 pH 8, 50 mM (kidne [41] PO50 4mM3− buffer Orn mM 50% at 10 Raty) 0.1 mM PLP, [14] 37 °C, pH mM (liver) 107.1, mM 50 Orn,mM 5 mM aKG PO43− buffer 47% at 25 Rat L-Valine Rat [6] 350.5 mM U OAT; Orn, 44%mM at 100 (liver) (kidne [41] 503.75 mM mM Orn mM aKG,K y) 37 °C, pH Rat 0.5 U OAT; 33% at 25 7.1, 50 mM (kidne [41] PO50 4mM3− buffer Orn 47%mM at 25 Rat L-Valine y) [6] pH35 mM 8, 50 Orn, mM mM (liver) Competitive 3.75 mM PO43− buffer 2.0 mM for Orn; Rat 0.1aKG,K mM PLP, (forward [14] uncompetiti (liver)Rat 100.5 mM U OAT; Orn, 33%reaction at 25) ve for aKG (kidne [41] 505 m mMM aKG Orn mM 37 °C, pH 8, y) pH 8, 50 mM Competitive 50 mM PO43− buffer 2.0 mM Noncompetifor Orn; Tris-HCl 20 mM Rat 0.1 mM PLP, (forward Rat [14] uncompetititive for Glu buffer 0.1 (reverse (liver) [14] 10 mM Orn, reaction) (liver) veand for P5CaKG mM PLP, 5 reaction) mM5 mM P5C, aKG 40 37 °C, pH 8, mM L-Glu pH50 8,mM 12.5 Noncompeti TrismM-HCl 28%20 mM at 2.5 Rat Competitivetive for Glu bufferTris-HCl 0.1 mM(reverse 41% at [14][23] (brain)(liver) and P5C buffermM PLP, 0.025 5 reaction5 mM ) mMmM P5C, PLP, 40 5 mM L-Glu pH 8, 12.5 mM 28% at 2.5 Rat Competitive Tris-HCl mM 41% at [23] (brain) buffer 0.025 5 mM mM PLP, 5

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Conditions α-Amino acids 8 U OAT, 37 °C, pH 8, 0.05 mM PO43− buffer Rat L-Canaline Irreversible Ki: 2 µM [39] 0.1 mM PLP, (liver) 175 mM Orn, 35 mM aKG Biology 2017, 6, 1 pH 8, 0.05 6 of 40

mM PO43− Irreversible, buffer 0.01 Rat noncompetit Conditions [40] mM PLP, 20 (liver) α-Amino acids ive 8mM U OAT, Orn, 1037 mM°C, pH aKG 8, 370.05 °C, mM pH PO7.1,43− 50 buffer mM Rat L i -Canaline Irreversible PO43− buffer 100%K : 2 atµM 25 Rat [39] L-Canavanine 0.1 mM PLP, (liver) [6] 35175 mM mM Orn, mM (liver) Orn,3.75 35 mM mM aKG,K aKG Rat 0.5pH U 8, OAT; 0.05 23% at 20

mM PO43− (kidne [41] Irreversible, 50 mM Orn mM buffer 0.01 Raty) noncompetit [40] mM37 °C, PLP, pH 20 (liver) ive mM7.1, 50Orn, mM 10 PO43− buffer 59% at 50 Rat L-Norvaline mM aKG [6] 3537 mM °C, Orn,pH mM (liver) 3.75 mM Biology 2017, 6, 1 7.1, 50 mM 7 of 40 POaKG,K43− buffer 100% at 25 Rat L-Canavanine pH 8, 50 mM [6] m35M mM Orn, Orn, 2.5 mM (liver) Biology 2017, 6, 1 PO43− buffer 7 of 40 3.75aKG mM 50% at 10 Rat 0.1 mM PLP, [14] Biology 2017 6 aKG,K , , 18 37 °C, pH mM (liver) 6 of 38 10m MmM Orn, Orn, 2.5 Rat 0.57.1, U 50 OAT; mM 23% at 20 5 mMaKG aKG (kidne [41] PO50 mM43− buffer Orn 27%mM at 25 Rat L-Isoleucine Rat [6] Table 2. Cont350.5.37 mM U °C, OAT; Orn, pH 44%mM at 100 (liver)y) (kidne [41] 50377.1,3.75 mM °C, 50 mM pHOrnmM mM y) 7.1,4 503− mM POaKG,KExperimental buffer 27% atK 25or % Rat OAT L-IsoleucineInhibitor Structure Nature 37 °C, pH i [6]Reference PO35 4mMConditions3− buffer Orn, 59%mM atInhibition 50 (liver)Rat Origin L-Norvaline pH 8, 50 mM [6] 357.1, mM37 50◦C, mMOrn, pH 7.1, mM (liver) 3.7543− mM PO 3− buffer 3− PO3.75504aKG,K mM buffermM PO 4 47%30% at 2510 Rat Rat L-ValineL -Norvaline 0.1 buffermM PLP, 35 mM 59% at 50 mM [14][6] [6] 35 mM Orn, mM (liver) (liver) 10pH aKG,KmMOrn, 8, 50 3.75Orn, mM mM 3.753− aKG,KmM pH5PO mM pH8,4 50 8,bufferaKG 50mM mM Biology 2017, 6, 1 aKG,K3− 3− 30% at 10 Rat 7 of 40 PO0.1 PO4mM 4buffer PLP,buffer Rat Rat [14] 0.5 0.1U OAT; mM PLP, 50%19%mM50% at 1025 at 10 mM(liver)Rat (liver) [14] 0.10.510 mM mM10U mMOAT; PLP,Orn, Orn, 33% at 25 (kidne [14][41] m50M mM 5Orn, mM Orn aKG2.5 mM (kidne(liver) [41] 10505 mMmM Orn,aKGOrn mM y) 0.5 U OAT; 44% at 100 mM Rat 5 mMaKG50 mM aKG Orn y)Rat (kidney) [41] pH 8,◦ 12.5 pH0.537 378, °C,U 50C, OAT; pH mM pH 7.1, 19% at 25 Competitive 3− (kidneRat [41] 0.55050 mMU3− mM OAT; Orn PO4 44%mM at 100 Rat L PO7.1,4 50 buffer mM 2.0 47%mM at 25 mM -Valine for Orn; buffer 35 mM (kidneRaty) (liver) [41] [6] 50Tris mM3− -HCl Orn 29%mM at 2.5 0.1PO Orn,mM4 buffer 3.75PLP, mM 27%(forward at 25 Rat [14] L-Isoleucine uncompetiti bufferpH 8,aKG,K 0.025 12.5 mM 29% at (liver)y) [23][6] Biology 2017, 6, 1 1035 mM0.5 UOrn, OAT; reactionmM ) (liver) Rat 7 of 40 37 °C,mM pH 33% at 25 mM(brain) ve for aKG mM50 PL mMP, Orn5 5 mM (kidney) [41] 53.75 mpHM mM 8,aKG 50 mM Competitive 7.1,Tris 50- HClmM 29% at 2.5 mmMM Orn,3− 2.5 Rat for Orn; 37 aKG,K°C,PO43− 4 pHbuffer 8, 2.0 mM Rat PObufferaKG buffer 0.025 mM47% 29%at 25 at Rat [23] [14] L-Valine uncompetitive pH50 8,0.1aKG mM50 mM mM PLP, (forward reaction(brain)) (liver) [6] for aKG 35mM37 mM 10°C, PL mM Orn,pHP, Orn, 5 5mM mM (liver) Noncompeti PO37Tris4 3−°C,5 mM-bufferHCl pH aKG 20 mM mM7.1,3.7537 50Orn, ◦mM C,mM pH 2.5 8, 30% at 10 Rat tive for Glu 0.17.1,buffer50 mM mM50 mMPLP,0.1 Tris-HCl (reverse [14] Noncompetitive aKG,K3−aKG 20 mM Rat PO43− buffer 21%mM at 25 (liver)Rat [14] forand Glu P5C and P5C 10POmM buffermM4 PLP, buffer Orn, 0.1 5 mM 27%reaction(reverse at 25) reaction)Rat (liver) L-LIsoleucine-Leucine 37PLP, °C, 5 mMpH P5C, Rat [6] 350.5 mM U OAT; Orn, 33%mM at 25 (liver) mM57.1, mM40 P5C, 50 mM aKG mM L40-Glu (kidne [41] 503.75 pHmM 8,mM 12.5Orn mM mM mM3.753− L mM-Glu Rat 0.5POTris-HCl U4 OAT;buffer buffer 19%21%28% at at 25 at25 2.5 mM y)Rat Rat L-Leucine Competitive 0.025aKG,K mM PLP, [6] [23] pH35pH 8,mM 8, 50 12.5 Orn,mM mM41% at 5 mM(kidne(liver)(brain) [41] Competitive pH50 mM8,5 mM50 Orn mM Orn, mM pH 8,3−mM 2.550 aKGmM 28% at 2.5 y) PO3.7543−◦ buffer mM 2.0 mM PO43− buffer Rat for Orn; POpH374 8, bufferC, 12.5 pH 7.1, Rat Competitive 0.1Tris mMaKG,K-HCl PLP, 3 − mM20%(forward 41% at 10 at Rat [14][23] 0.1 50mM mM PLP, PO4 30% at 10 (brain)Rat Rat [14] L -Isoleucine uncompetiti 0.1 mMmM PLP, 27% at 25 mM(liver) [14] [6] 10bufferpH mMbuffer 8, 500.025 Orn, 35mM mM reaction5mM mM ) (liver) (liver) ve for aKG 10 mMOrn, 3.75Orn, mM 10mMTris mM 3−PLP,-HCl Orn, 5 29% at 2.5 5PO mmM4MaKG,K bufferaKG Rat buffer5 mMpH 8,0.025aKG 50 mM mM20% 29% at 10 at Rat [23] 370.1 °C, mM pH3− PLP, 8, (brain) [14] PO4 buffer mM (liver)Rat Rat mM0.5 U PL OAT;P, 5 17%5 mM30% at 25 at 10 mMRat [14] 0.51050 mM0.1U mM OAT; mM Orn, PLP, 19% at 25 (kidne (liver) [41] mM50 mM10 Orn, mM Orn 2.5 Orn, mM (kidne [41] Noncompeti 505Tris mM5 mM-HCl aKGOrn aKG 20mM mM y) aKG0.5 U OAT; Raty) Rat tive for Glu buffer 0.1 (reverse19% at 25 mM Rat [14] [41] pH0.550 U8, mM OAT;12.5 Orn 17% at 25 (liver) (kidney) and P5C mM37pH °C, PLP, 8, 12.5pH 5 mM reaction) (kidne [41] 50 mM Orn mM 7.1,Tris-HCl 50 mM buffer 29% at 2.5 mM Rat mMTris0.025 P5C,-HCl mM 40 PLP, 8% at 2.5 y) [23] POTris43− -bufferHCl 29%21% at29%at 2.525 at 5 mM Rat (brain) mMpH5 8,L mM- Glu12.5 Orn, Rat L-Leucine buffer2.5 0.025 aKG mM 22% at [23][6] 35buffer mM◦ 0.025 Orn, mMmM 29% at (brain)(liver) [23] mMpH37 mM 8,PLP,C, 12.5 pH 5 7.1, 5 mM (brain) mM3.75 PL mMP, 5 3− 5 mM Tris50mM mM-HCl PO 4 28%8% at 2.5 Rat L-Leucine mM Orn, 2.5 21% at 25 mM mMaKG,Kbuffer Orn, 35 2.5 mM RatRat (liver) [6] Competitive bufferTrisOrn,-HCl 3.750.025 mM mMmM 41% 22% at at [23][23] aKG (brain)(brain) pHbuffermM 8, 50aKG,KPLP, 0.025 mM 5 55 mM mM pHpH3− 8, 8,12.5 50 mM PO374 °C, buffer3 −pH mMPO Orn,PLP,4 buffer 2.55 20% at 10 Rat Rat 7.1, mM50 mM 20% at 10 mM [14] 0.1 mM0.1aKG mM PLP, PLP, (liver) [14] Tris103− - mMHCl Orn, 48%mM at 2.5 (liver) 10PO mM4 buffer Orn, 21% at 25 Rat L-Leucine pH5 mM8, 12.5 aKG Rat [6] Cysteine buffer0.5 0.025 U OAT; mM 66% at Rat [23] 355 mM mM aKG Orn, mM17% at 25 mM(brain)(liver) [41] mM50mM PLP, mM Orn5 5 mM (kidney) 3.75pH 8,mM 12.5 mM Rat mM0.5Tris U Orn, OAT;-HCl 2.5 17%48% atat 252.5 Tris-HClaKG,K buffer 8% at 2.5 mM(kidneRat Rat [41] Cysteine 50buffer 0.025mMaKG mM0.025Orn PLP, mMmM 66%22% at at 5 mM (brain) [23] [23] pH 8,5 mM50 mM Orn, (brain)y) mM 2.5PLP, aKG 5 5 mM PO37 °C,43− buffer pH 8, mMpHpH 8,Orn, 8, 12.5 12.5 2.5 mM 20% at 10 Rat 0.150 Tris-HCl mMmM POPLP, buffer43− 25%48% at 10 at 2.5 mM Rat [14] Cysteine mM 0.025aKG mM PLP, mM66% at 5 mM(liver)Rat (brain) [23] 2,4-Diaminobutyrate Competitive 10bufferTris mM5- mMHCl Orn,175 Orn, mM8% 40%at 2.5 at [42] (liver)Rat mM537 mM °C, Orn,2.5 aKGpH aKG 35 8, 25 mM buffer◦ 0.025 mM 22% at [23] 50 mM37 C, PO pH43− 8, 25% at 10 (brain)Rat mMmM PLP, aKG 5 3− 5 mM25% at 10 mM Rat Rat 2,4-Diaminobutyrate2,4-Diaminobutyrate CompetitiveCompetitive 0.5buffer50 U mM OAT; 175 PO4 mM17% 40%at 25 at [42] [42] γ-Amino acids mMbuffer Orn, 175 2.5 mM 40% at 25 mM(kidne(liver) (liver) [41] 50mMOrn, mM Orn, 35 Orn mM 35 aKG 25mM mM aKG y) γ-Amino acids 37 °C, pH 8, pHmM 8,◦ aKG12.5 3.4 mM 50pH mM37 8, C,12.5PO pH4 3− 8, 3− 70%3.4 at mM 10 70% at 10Rat Rat γ-Aminoγ acids Competitive 50mM mM PO4 mM 90% at 25 γ-Aminobutyrate-Aminobutyrate Competitive buffer 175 (liver) [42] [42] Trisbuffer-HCl 175 mM mM8% 90%at 2.5 mMat (liver) mM37Orn,Tris °C, Orn,- 35HCl pH mM 35 8, aKG 48% at 2.5 ◦ 3.4 mM Rat Cysteine buffer50 37mMC, 0.025 PO pH4 7.1,3− mM25 66%22%mM at [23] mM aKG 3− 70% at 10 (brain)Rat 50 mM PO4 (brain) Rat γ-Aminobutyrate Competitive mMbuffer PLP, 175 5 5 mM47% at 25 mM [42] 37buffer °C, pH 35 mM mM 90% at (liver) (liver) [6] mMmMOrn, Orn, Orn, 3.75 2.5 35 mM 47% at 25 Rat 7.1, 50aKG,K mM 25 mM [6] mMpHaKG 8,aKG50 mM mM (liver) PO4aKG3− buffer 3− pH37PO °C,8,4 12.5 pHbuffer Rat 26% at 10 mM 37 °C,0.1 mMpH PLP,8, 47% at 25 Rat (liver) [14] 7.1,10mM 50 mM mM Orn, [6] 50 mM PO43− 25%mM at 10 (liver) 53− mM aKG Rat POTris40.5- HClbuffer U OAT; 48% at 2.5 Rat 2,4-Diaminobutyrate Competitive buffer 175 mM 40%26% atat 25 mMRat [42] Cysteine(GABA) buffer50 mM0.025 Orn mM 66% at (liver) (kidney)[23] [41] mM Orn, 35 25 mM (brain) mM PLP, 5 5 mM mM aKG mM Orn, 2.5 γ-Amino acids aKG 37 °C, pH 8, 37 °C, pH 8, 3.4 mM 50 mM PO43− 50 mM PO43− 70%25% at 10 Rat γ-Aminobutyrate Competitive buffer 175 Rat [42] 2,4-Diaminobutyrate Competitive buffer 175 mM 90%40% at (liver) [42] mM Orn, 35 (liver) mM Orn, 35 25 mM mM aKG mM aKG 37 °C, pH 47% at 25 Rat γ-Amino acids 7.1, 50 mM [6] mM (liver) 37 °C,3− pH 8, PO4 buffer 3.4 mM 50 mM PO43− 70% at 10 Rat γ-Aminobutyrate Competitive buffer 175 [42] mM 90% at (liver) mM Orn, 35 25 mM mM aKG 37 °C, pH 47% at 25 Rat 7.1, 50 mM [6] mM (liver) PO43− buffer

Biology 2017, 6, 1 8 of 40

35 mM Orn, Biology 2017, 6, 1 3.75 mM 8 of 40 aKG,K pH35 mM8, 50 Orn,mM PO43− buffer 3.75 mM 26% at 10 Rat 0.1 mM PLP, [14] Biology 2017, 6, 1 aKG,K mM (liver) 8 of 40 pH10 mM 8, 50 Orn, mM 5 mM aKG PO43− buffer 35 mM Orn, 26% at 10 RatRat 0.10.5 mMU OAT; PLP, 26% at 25 [14] (GABA) 3.75 mM mM (kidne(liver) [41] 5010aKG,K mM OrnOrn, mM Biology 2017, 6, 1 y) 8 of 40 pH5 mM8, 50 aKG mM 37 °C, pH Rat PO43− buffer 7.1,0.5 U50 OAT; mM 26%26% at at 10 25 Rat (GABA) 0.135 mMmM Orn,PLP, (kidne [14][41] PO50 4mM3− buffer Orn 100%mMmM at 25 (liver)Rat Biology5-Aminovalerate 2017, 6, 1 103.75 mM mM Orn, y) 8 [6]of 40 3537 aKG,KmM °C, Orn,pH mM (liver) 5 3.75mM mMaKG pH357.1, mM8, 5050 Orn, mMmM aKG,K Rat PO0.5PO3.754 4U3−3− buffer OAT;mMbuffer 26%100% at at 25 25 Rat 5-Aminovalerate(GABA) 26% at 10 (kidneRatRat [41][6] 0.150350.5 aKG,K mMmM U OAT; PLP,OrnOrn, 15%mMmM at 25 (liver) [14] Biology(δ-Aminovalerate) 2017, 6, 1 mM (liver)(kidney) 8[41] of 40 pH1050 3.75mM mM8, 50 mM Orn, OrnmM mM 37 °C, pH y) PO5 mM4aKG,K3− buffer aKG 7.1,35 mM 50 mM Orn, 26% at 10 Rat pH 8, 0.05 RatRat 0.1PO mM43− buffer PLP, 100% at 25 Rat [14] 0.50.5mM3.75 U U POOAT; OAT;mM43− 26%15%mM at at 25 25 (liver) (δ5--AminovalerateAminovalerate)(GABA) 10 mM Orn, (kidne(kidne [41][6][41] Competitive 355050buffer mM aKG,KmM Orn,0.01Orn Orn mMmM (liver)Rat 5 mM aKG 17.7 mM y)y) [40] for Orn mMpH3.75 8, PLP, 50mM mM 20 (liver) 37pH °C, 8, pH0.05 Rat mM0.5POaKG,K 4U 3−Orn, OAT;buffer 10 26% at 25 (GABA) 7.1,mM 50 PO mM43− 26% at 10 (kidneRat [41] 0.150mM mMmM aKG OrnPLP, mM Rat [14] Competitive PO0.5buffer4 U3− bufferOAT; 0.01 100%15%mM atat 25 25 (liver)RatRaty) (δ5-AminovalerateAminovalerate) 10 mM Orn, 17.7 mM (kidne [41][6][40] for Orn 3550mM37 mMmM °C, PLP, Orn,pHOrn 20 mM (liver)(liver) 5 mM aKG y) mM7.1,3.75 50 Orn, mM mM 10 pH 8, 0.05 RatRat Biology 2017, 6, 18 3− 7 of 38 PO0.50.5mMaKG,K4 UU buffer OAT;aKGOAT; 3− 100%26%6% at at 25 25 Rat ε5--AminocaproateAminovalerate(GABA) mM PO4 (kidne(kidne [41][6][41] 5050 mMmM OrnOrn mMmM Rat Competitive 35buffer mM 0.01Orn, mM (liver)Raty) 0.5 U OAT; 15%17.7 atmM 25 y) [40] (δ-Aminovalerate) for Orn mM3.75 PLP, mM 20 (kidne(liver) [41] Table 2. Cont50.37 mM °C, Orn pH mM Rat mM0.5aKG,K UOrn, OAT; 10 6% at 25 y) ε-Aminocaproate 7.1, 50 mM (kidne [41] α-Ketoacids pH 8, 0.05 Rat PO50mM mM43− aKGbuffer Orn 100%mM at 25 Rat 0.5Experimental U OAT;3− 15% atK 25or % y) OAT (δ5--Aminovalerate)AminovalerateInhibitor Structure Nature mM37 °C, PO pH4 i (kidne [41][6]Reference 5035 mMmMConditions OrnOrn, mMmMInhibition (liver)Origin Competitive buffer7.1, 50 0.01mM Raty) 3.7537 ◦C, mM pH 7.1, 17.7 mM [40] Biology 2017, 6, 1 for Orn mMPOpH4 3− PLP,8, buffer 0.05 20 3 − 66% at 21 (liver)Rat 9 of 40 α-Ketoacids 50 mM PO4 Rat α-Ketovalerate5-Aminovalerate 0.5 aKG,KU OAT; 6%100% at 25 at 25 mM [6] [6] ε-Aminocaproate mM35mM buffermM Orn, PO Orn, 354 3−10 mM mM (kidne(liver) (liver) [41] 5037 Orn,mM °C, 3.75 OrnpH mM mM Rat Competitive buffer0.5mM3.75aKG,K U aKG,K aKG mMOAT; 0.01 15% at 25 Raty) (δ-Aminovalerate) 7.1,0.5 50 U mM OAT; 17.7 mM (kidne Rat [40][41] δ 15% at 25 mM ( -Aminovalerate) for Orn mM50aKG,K 50mM 3−PLP, mM Orn Orn20 mM (liver)Rat (kidney) [41] 0.5PO U4 OAT; buffer 34%66% at at 25 21 Raty) α-Ketovalerate mMpH Orn, 8, 0.05 10 mM (kidneRat [41][6] 350.5pH mM U 8, 3OAT;− 0.05Orn, 38%mM at 25 (liver) α-Ketoacids Competitive 50 mMPO4 Ornbuffer mM Rat Rat 0.5mM0.01 U OAT;aKG mM3− PLP, 6% at 17.725 mM (kidney) [41] [40] ε-Aminocaproate for Orn 5037mM3.75 mM °C, POmM pHOrn4 mM (kidne (liver) [41] 50 mM20 mM Orn Orn, mM y) Competitive 37buffer aKG,K10°C, mM pH 0.01 aKG Rat 7.1, 50 mM 17.7 mM y) [40] 7.1,37 °C,50 mM pH Rat for Orn POmM43− PLP, buffer 20 66% at 21 (liver)Rat 7.1,0.53− U50 OAT; mM 38% at 25 Rat α-Ketovalerate PO0.5mM4 U0.5 Orn, bufferOAT; U OAT; 10 13%6% atat 2510 (kidneRat Rat [6][41] εα-Aminocaproate-Ketεobutyrate-Aminocaproate 3550 mM mM3− Orn, Orn mMmM6% at 25 mM(kidne(liver) [41][6] [41] α-Ketoacids 35PO50 mMmM450 mMbuffer Orn,Orn Orn 60%mM at 19 (liver)Raty) (kidney) α-Ketoisocaproate 3.75mM mMaKG y) [6] 35373.7537 mM °C, °C, mM pH Orn,pH mM (liver) α-Ketoacids 3.75aKG,K mM 7.1,aKG,K 50◦ mM 7.1,37 50C, mM pH 7.1, Rat 0.5aKG,K U3−3− OAT; 3− 38% at 25 RatRat α-Ketoacids POPO5044 mM buffer buffer PO4 66%60% at at 21 19 RatRat Rat αα-Ketoisocaproate-Ketovalerateα 0.50.5 U U OAT; OAT; 27%6%66% atat 2525 at 21 mM(kidne [41][6][6] ε-Aminocaproate -Ketovalerate 50 buffermM Orn 35 mM mM (kidne(kidneRat (liver) [41][41] [6] 35350.55037 Orn,mM mM mMU°C, OAT;3.75 Orn, pH Orn,Orn mM 41%mMmM at 25 (liver)(liver) (Ketoleucine) 50 mM Orn mM (kidney) [41] 7.1,3.753.75 50aKG,K mM mM mM y)y) 5037 mM 0.5°C, U pHOrn OAT; mM Rat 38% at 25 mM y) [41] Polyamines POaKG,K4aKG,K503− mMbuffer Orn 66% at 21 Rat (kidney) α-Ketovalerate 7.1, 50◦ mM [6] 373737 °C,°C,C, pHpH pH 7.1, RatRat 35 mM43− Orn,3 − mM (liver) α-Ketoacids PO0.50.550 U U mM bufferOAT; OAT; PO4 38%60%41% at at 2519 25 Rat Rat α-Ketoisocaproateα-Ketoisocaproate 7.1,7.1, 5050 mMmM 60% at 19 mM(kidne [41][6] [6] (Ketoleucine) 353.75 buffermM mM Orn, 35 mM mM (liver)(kidne (liver) [41] 50PO5037 Orn,mM 43−mM 3−°C, buffer 3.75 Orn pHOrn mM 45%mMmM at 22 Rat POaKG,K4 buffer 14% at 25 Raty)y) α-KetoisovalerateCadaverine 7.1,3.75 50aKG,K mM mM [6][6] 35 mM0.5 U Orn, OAT; mM (liver) Rat (Ketoleucine) 353737 mM °C, °C, Orn,pH pH mM41% at 25 mM(liver)Rat [41] POaKG,K5043− mMbuffer Orn 66% at 21 Rat (kidney) H2N-(CH2)5-NH2 0.53.753.75 U◦ mMOAT;mM 38% at 25 α-Ketovalerate 7.1,7.1,37 50 50C, mM mM pH 7.1, (kidneRat [41][6] 5035 aKG, mMmM OrnKOrn, 3 − mMmM (liver) PO0.5POaKG,K504 4U3−3− mM buffer OAT;buffer PO 4 60%41%45% at at 1925 22 RatRat Rat (Ketoleucine)α-Ketoisovalerate 45% at 22 mM(kidney) [41] [6] αα-Ketoisocaproate-Ketoisovalerate 3.75buffer mM 35 mM Rat (liver) [6][6] Biology 2017, 6, 1 35503537 Orn,mM mMmM °C,3.75 Orn, pHOrnOrn, mM mMmM (liver)(liver)Rat 9 of 40 0.50.5 aKG,K UU OAT;OAT; 5.7%34% at 25 y) aKG,K (kidne(kidne [41][41] 507.1,3.753.75 mM0.5 50 mM UmMmM Orn OAT; mM Rat 5037 mM °C, OrnpH mM34% at 25 mM Rat [41] 503− mM Orn y)y) (kidney) PO0.5aKG,KaKG,K4aKG, U◦ buffer OAT;K 60%38% at at 19 25 Rat α-Ketoisocaproate 7.1,3737 °C,50C, mMpH pH 7.1, (kidne [6][41] 5037 mM°C, pH Orn 3 − mM RatRat 35PO 50mM43− mM buffer Orn, PO4 45%mM at 22 (liver)RatRat α-Ketocaproate 0.50.57.1,0.5 UU U50 OAT;OAT; OAT; mM 34%41%34% 18% atat at 2525 at25 15 mM y) Rat α-(Ketoleucine)Ketoisovalerate 7.1,3.75buffer 50 mM mM 35 mM 18% at 15 (kidne(kidne(kidneRat (liver)[41][41][6][41] [6] 35505037 Orn,mM mM 3−°C, 3.75 Orn,Orn pHOrn mM mMmM (liver) α-Ketocaproate 50POPO mM443− bufferbuffer Orn 14%mM at 25 Rat [6] 3.75aKG,KaKG,K mM mM (liver)y)y)y) Putrescine 357.1, mM0.5 50 U Orn,mM OAT; Rat [6] 353737 mM °C, °C, Orn,pH pH mM34% at 25 mM(liver) [41] 37aKG, °C,503− mM pHK Orn Rat (kidney) 0.5PO3.75 4U◦ OAT;mMbuffer 41%60% at at 25 19 Rat α-(Ketoleucine)Ketoisocaproate 7.1,7.1,7.1,3.7537 5050 50 C,mM mMmM mM pH 7.1, (kidne [41][6] 35 mM Orn,3 − 18%mM at 15 (liver)RatRat 500.550 mM U3−3− mM OAT; Orn PO4 34%mM at 25 Rat α-Ketocaproateα POPOPOaKG,K443−4 bufferbuffer buffer 13%45% 13% atat 1022 at 10 mMRatRaty) [6] α-Ketoisovalerate-Ketobutyrate 3.75buffer mM 35 mM mM (kidne(liver)(liver) [41][6] [6] α-Ketobutyrate pH3550 Orn,mM8,mM 50 3.75 Orn, OrnmM mM mM (liver) [6] 353537 mM mM °C, Orn, pHOrn, mM (liver)y) aKG,K3− aKG,K PO3.753.754 0.5 buffermM UmM OAT; No Rat 7.1,3.75 50 mM mM 27% at 25 mM 37 50°C, mM pH Orn RatRat (kidney) [41] 0.1PO0.5 aKG,mM4 3−U buffer OAT; PLPK , inhibition45%41% at at 22 25at Rat [14] (Ketoleucine)Polyamines 7.1,aKG,K 50 mM (liver)(kidne [41] α-Ketoisovalerate 10 mM◦ Orn, 18%10 mM at 15 Rat [6] 3550 37mM mM3− C, Orn, pHOrn 7.1, mMmM (liver)RatRat α-Ketocaproate 0.5PO0.5 4UU OAT;bufferOAT; 3− 27%34% atat 2525 y) [6] H2N-CH2-CH2-CH2-CH2-NH2 5 mM50 mM aKG PO4 mM (kidne(liver) Rat [41] Cadaverine 353.75 buffermM mM Orn, 35 mM 14% at 25 mM(kidne [41] [6] H2N-(CH2)5-NH2 505037 mMmM °C, OrnOrn pH mMmM (liver) Orn,aKG, 3.75K mM Raty)y) 0.57.1,3.75 U 50 aKG,K OAT;mM mM 3% at 25 37 °C,0.5 U pH OAT; (kidne Rat [41] Polyamines 50 mM3− Orn mM5.7% at 25 mMRat 0.5PO 504U mMOAT;buffer Orn 34%45% at at 25 22 Rat (kidney) [41] α-Ketoisovalerate 7.1,37 °C, 50◦ pHmM (kidney) [41][6] 5035 37 mMmMC, OrnOrn, pH 7.1, 18%mMmM at 15 (liver)Rat α-Ketocaproate POpH43− 8, buffer 12.5 3− [6] 7.1,50 50 mM mM PO 4 y) Rat Putrescine 3.75buffer mM 35 mM mM14% at 25 mM(liver) [6] 35 mM3−mM Orn, (liver) PO37Orn,4 °C, buffer3.75 pH mM 14%No at 25 Rat Cadaverine H2N-CH2-CH2-CH2-CH2-NH2 aKG,K [6] 357.1,Tris3.75 mM 50-aKG,K HClmM Orn,mM mM (liver) pH 8, 50 mM inhibition18% at 15 at RatRat α-Ketocaproate H2N-(CH2)5-NH2 bufferPO0.53.754 3−U mMbuffer 30.025OAT;− 34% at 25 [23][6] PO4 buffer 5 mMNo inhibition 8% at(brain)(kidne 10 Rat [41] mM (liver) [14] 35mM50aKG,K mM 0.1mM PLP, mM Orn, Orn PLP,5 mM mM (liver) 10 mM Orn, at 10 mM y) mM3.75 5Orn, mM mM aKG2.5 Rat 0.537 U0.5 °C, OAT; U pH OAT; 5.7% at 25 Rat aKG 3% at 25 mM(kidne [41] [41] 507.1, mM50 50 mM Orn mM Orn mM (kidney) pHpH 8, 8, 12. 12.55 mM 18% at 15 y)Rat α-Ketocaproate POTris-HCl43− buffer buffer [6] 37 °C,mM pH NomM inhibition at(liver) 5 Rat 350.025 mM mM Orn, PLP, mM 8% at 10 mM (brain) [23] 7.1,Tris 550 mM-HCl mM Orn, 33% at 5 3.752.5 mM aKG Rat Spermine buffer 0.025 mM 56% at [23] POpH43− 8,buffer 12.5 mM 14% at 25 (brain)Rat Putrescine mMTris-HCl PLP, buffer5 1033% mM at 5 mM 56% Rat [6] Spermine 35 mM Orn, mM (liver) 0.025 mM PLP, at 10 mM (brain) [23] mM3.75 5Orn, mMmM Orn,2.5 2.5 aKG aKG,KpHaKG 8,50 mM 3− pH 8,PO 504 mMbuffer No inhibition at 10 Rat 0.1 mM PLP, [14] 43− mM (liver) PO4103− buffer mM Orn, No 5 mM aKG Rat 0.1 mM PLPPLP,, inhibitioninhibition at [14][14] (liver)(liver) 10 mM Orn, 10 mM H2N-CH2-CH2-CH2-CH2-NH2 5 mM aKG pH 8, 12.5 Rat 0.5 U OAT; 3% at 25 mM (kidne [41] 50Tris mM-HCl Orn 30%mM at 5 Raty) Spermidine buffer 0.025 mM 48% at [23] pH 8, 12.5 (brain) mMmM PLP, 5 10 mM No mMTris Orn,-HCl 2.5 inhibition at Rat bufferaKG 0.025 [23] 5 mM 8% (brain) mM PLP, 5 at 10 mM mM Orn, 2.5 aKG pH 8, 12.5 mM Tris-HCl 33% at 5 Rat Spermine buffer 0.025 mM 56% at [23] (brain) mM PLP, 5 10 mM mM Orn, 2.5 aKG

pH 8, 50 mM PO43− buffer No Rat 0.1 mM PLP, inhibition at [14] (liver) 10 mM Orn, 10 mM 5 mM aKG pH 8, 12.5 mM Tris-HCl 30% at 5 Rat Spermidine buffer 0.025 mM 48% at [23] (brain) mM PLP, 5 10 mM mM Orn, 2.5 aKG

Biology 2017, 6, 1 9 of 40

aKG,K Rat 0.5 U OAT; 34% at 25 (kidne [41] 50 mM Orn mM y) 37 °C, pH 7.1, 50 mM PO43− buffer 13% at 10 Rat α-Ketobutyrate [6] 35 mM Orn, mM (liver) 3.75 mM aKG,K Rat 0.5 U OAT; 27% at 25 (kidne [41] 50 mM Orn mM y) Polyamines 37 °C, pH 7.1, 50 mM PO43− buffer 14% at 25 Rat Cadaverine [6] 35 mM Orn, mM (liver) H2N-(CH2)5-NH2 3.75 mM aKG,K Rat 0.5 U OAT; 5.7% at 25 (kidne [41] 50 mM Orn mM y) 37 °C, pH 7.1, 50 mM PO43− buffer 14% at 25 Rat Putrescine [6] 35 mM Orn, mM (liver) 3.75 mM aKG,K pH 8, 50 mM PO43− buffer No Rat 0.1 mM PLP, inhibition at [14] (liver) 10 mM Orn, 10 mM H2N-CH2-CH2-CH2-CH2-NH2 5 mM aKG Rat 0.5 U OAT; 3% at 25 (kidne [41] 50 mM Orn mM y) pH 8, 12.5 mM No Tris-HCl inhibition at Rat Biology 2017 , 6, 1 buffer 0.025 10[23] of 40 5 mM 8% (brain) Biology 2017, 6, 1 mM PLP, 5 10 of 40 at 10 mM mMpH Orn,8, 12.5 2.5 pHaKGmM 8, 12.5 Biology 2017, 6, 1 pHTris 8,-HCl 12.5 42% at 5 10 of 40 mM Rat Histamine bufferTrismM-HCl 0.025 mM42% 50% at 5 at [23] (brain)Rat Histamine buffermMpHTris 8,PL- HCl 0.02512.5P, 5 mM33%10 50%mM at 5 at [23] (brain)Rat Spermine mMbuffermM mMOrn, PL 0.025P, 2.5 5 mM10 56%mM at [23] (brain) Biology 2017, 6, 1 TrisaKG-HCl 42% at 5 10 of 40 Biology 2017, 6, 18 mMmM Orn, PLP, 2.5 5 10 mM Rat 8 of 38 Histamine buffer 0.025 mM 50% at [23] Other inhibitors mMaKG Orn, 2.5 (brain) Other inhibitors mMpHaKG 8,PL 12.5P, 5 10 mM Rat 37 °C, pH 8, pHmM 8, mMOrn, 50 mM 2.5 (liver;Rat 5037 mM°C, pH PO 8,43− Biology5-Fluoromethylornithin 2017, 6, 1 POTris4aKG3− -bufferHCl 42%No at 5 partiall(liver; 10 of 40 Table 2.50buffer ContmM PO 175. 43− 70 µM Rat [37] 5-FluoromethylornithinOtherHistamine inhibitorse 0.1buffer mM 0.025 PLP, inhibitionmM 50% at partially [14][23] mMbuffer Orn, 175 35 70 µM (brain)(liver) [37] 10mMpH mM 8,PL 12.5Orn,P, 5 10 mM purifieRat e 37mM °C, aKGpH 8, y mM Orn, 35 (liver; mM5 mMmM Orn, aKG 2.53−Experimental purified) K or % OAT Inhibitor Structure Nature 50mM mM aKG PO4 i Reference 5-Fluoromethylornithin pH8Tris UaKG 8,OAT,-HCl 12.5 Conditions42% at 5 partialld) Inhibition Origin buffer 175 70 µM Rat [37] OtherHistamine inhibitorse buffer37°C,8 UmM OAT, pH0.025 8, mM 50% at y [23] mM Orn, 35pH 8, 12.5 mM (brain) Biology 2017, 6, 1 Irreversible 37°C,mMTris0.05 PL- pHHClmMP, 8,5 30%10 mM at 5 purifieRat 10 of 40 37mM °C, aKGpH 8,Tris-HCl buffer Rat30% at 5 mM 48% Rat SpermidineSpermidine Irreversible mMbufferPO0.054 3−Orn, buffermM 0.025 2.5 0.025 mM mM 48% PLP, at (liver;Ratd) [23] [23] 50 mM PO43− at 10 mM (brain) 8 U OAT, 30 µM (brain) [39] 5-Fluoromethylornithin 0.1POmMpH mM4aKG3− 8,PLP, buffer 12.5 PLP, 5 5 mM10 Orn,mM partiall(liver)Rat buffer 175 3070 µM [39][37] Other inhibitorse mM37°C,175 Orn, mMpH 2.58, 2.5 aKG y 0.1mM mMmM Orn, PLP, 35pH 8, 12.5 mM (liver) Irreversible Orn,0.05aKG 35 mM mM purifie mMTris175 -mMaKGHCl Tris-HCl 42% buffer at 5 Rat 37 °C,43− pH 8, Rat42% at 5 mM 50% Rat HistamineHistamine Orn,PObufferaKG 35 buffer 0.025 mM 0.025 mM mM 50% PLP, at (liver;Ratd) [23] [23] 50 mM PO43− 30 µM (brain) at 10[39] mM (brain) 5-Fluoromethylornithin 0.1mM8Not mMUaKG PLOAT,given PLP,P, 5 5 mM1110 ± Orn, mM2 µM partiall(liver) [36] buffer 175 70 µM [37] S,S-5-Fluoromethylornie 37°C,175 mMpH 8, 2.52.4 aKG ± 0.3 y mMNot Orn, given 2.5 11 ± 2 µM [36] Other inhibitors mMOrn, Orn,35 mM 35 S,S-5-Fluoromethylornithine Irreversible 0.05aKG mM ◦ 2.4µM ± 0.3 purifie mMNot givenaKG 37 C, pH 8, [36] Rat POpH4aKG3− 8, buffer 0.05 Rat Otherthine inhibitors 30µM µM 3 − d) [39] (liver; 3−50 mM PO4 µ 5-Fluoromethylornithine 0.1pHmM8Not UmM 8,OAT, givenPO 0.05 PLP,4 11 ± 2 µM (liver)Rat 70 [36]M partially [37] 37 °C, pH 8,buffer 175 mM S,S-5-Fluoromethylorni 175 mM3− 2.4 ± 0.3 CompetitiveIrreversible 37°C,buffermMNot given POpH 0.014 Orn,8, 35 mM aKG (liver;Rat [36] purified) N-Acetylornithine 50 mM PO43− 8.8 mM◦ [40] 5-Fluoromethylornithinthine CompetitiveIrreversiblewith Orn mMOrn,buffer0.05 PLP,35 mM 0.01 mM 20 8 U OAT,µM 37 C, partiall(liver)Rat N-Acetylornithine pHbuffer3− 8, 0.05 175pH 8, 0.058.870 mMµM mM [40][37] e with Orn mMPO4aKG PLP,Orn, buffer 2010 (liver)Raty mM Orn, 3−35 3−30 µM [39] Rat 0.1mMmMNot mM givenPOaKG PLP,4 PO 4 11 ±buffer 2 µM (liver) 30 µ[36]M mM Orn, 10 purifie (liver) [39] S,S-5-Fluoromethylorni Competitive bufferpHmM 8, aKG 12.50.01 0.1 mM2.4 ± PLP, 0.3 Rat N-Acetylornithine mMNot175 givenmMaKG 175 mM8.8 mM Orn, d) [40][36] thine with Orn Orn,mMpH8 U mM PLP,8, 35OAT, 12.5 mM 20 35 mMµM aKG (liver) mMpHTrisaKG Orn,8,-HCl 0.05 10 Not24% given at 2.5 11 ± 2 µM [36] 37°C,mM pH 8, Rat S,S-5-Fluoromethylornithine Irreversible buffermMNotTris0.05 given- POaKGHCl mM0.02543− NotmM24%11given ± 27%2at µM 2.5 at 2.4 ± 0.3[36][23]µ M [36] pH 8, 12.5pH 8, 0.05 mM (brain)Rat S,S-5-Fluoromethylorni Competitive bufferPOmMbuffer43− PLP, buffer 0.025 0.01 5 mM2.45 mM27%± 0.3 at Rat [23] N-Acetylornithine Not given 3−8.8 mM (brain) [36][40] withCompetitive Orn mM mMOrn,PLP, 2.520PO 4 30buffer µM (liver) [39] Rat N-Acetylornithinethine 0.1mM mM PLP, PLP, 5 5µM mM (liver) 8.8 mM [40] with OrnmMTris Orn,-HCl 10 0.01 24% mM at PLP, 2.5 (liver) mMpH175aKG Orn,8, mM 0.05 2.5 20 mM Orn, Rat buffer0.5mMmM aKGU POOAT, aKG0.025 43− mM 27% at [23] Orn, 35 mM 10 mM aKG (brain) Competitive 0.5mMbufferpH37 UaKG °C,8,PLP, OAT, 12.50.01 40 5 pH 8, 12.55 mM mM Rat N-Acetylornithine 8.8 mM [40] with Orn mMmMNot37 mMOrn,°C,PLP, given KPP 40 2.520 Tris-HCl 11 ± buffer2 µM (liver) 24% at 2.5[36] mM Rat Rat [23] 4-Aminohexynoate Irreversible mM0,020TrisaKG Orn,- HClmM 10 0.025 24% mM at PLP, 2.5 27% at[43] 5 mM (brain) S,S-5-Fluoromethylorni mM KPP 5 mM2.4 Orn, ± 0.3 (liver)Rat PLP,buffer0.5Not U 60 givenOAT, 0.025 mM mM 27% at Rat [36][23] 4-Aminohexynoatethine Irreversible 0,020mM aKG mM 2.5 aKGµM (brain) [43] Orn,mM37 °C, 21PLP, mM40 5 5 mM (liver) PLP,pH 8,60 12.50.05 mM ◦ mM KPP0.5 U OAT, 37 C, mMOrn,mMaKGmM Orn,21 PO mM 4 2.53− 40 mM KPP0,020 Rat Rat 4-Aminohexynoate4-Aminohexynoate CompetitiveIrreversibleIrreversible 0.5buffer0,020Tris aKGU- HClOAT, mM 0.01 24% at 2.5 Rat [43] [43] N-Acetylornithine mM PLP,8.860 mM mM (liver)Rat [40] (liver) with Orn PLP,mMbuffer0.537 U °C,PLP,60 OAT, 0.025 mM40 Orn,20 21mM mM 27% aKG at (liver) [23] (brain) Orn,mMmM37 °C,21PLP, KPP mM40 5 5 mM mM37 °C,Orn, 40 10 Rat Gabaculine Irreversible mM0,020mM aKGOrn, KPP mM 2.50.5 U OAT, 37 ◦C, [43] mM KPPaKG (liver)Rat 4-Aminohexynoate Irreversible PLP,0.50,020 U 60 OAT, mM mM40 mM KPP 0,020 Rat [43] Rat GabaculineGabaculine IrreversibleIrreversible 0,020pHaKG 8, mM12.5 (liver) [43] [43] Orn,PLP,37 °C, 2160 mM40mM PLP, 60 mM (liver) (liver) PLP,0.5 UmM 60 OAT, mM Orn, 21 mM aKG Orn,mMaKG 21 KPP mM Orn,37Tris °C, 21-HCl mM40 24% at 2.5 Rat Gabaculine Irreversible 0,020 mM Rat [43] buffermMaKG KPP 0.025 mM“almost 27% at [23] (brain)(liver)Rat “almost 2-aminooxyacetate4-Aminohexynoate Irreversible PLP,0.5mM0,020 U 60PLP, OAT,mM mM 5 5 mM complete”[43] Rat [44] 2-aminooxyacetate complete”“almost (liver)Rat [44] Orn,37 °C,21 mM40 at 0.2 mM 2-aminooxyacetate mMPLP, Orn,60 mM 2.5 complete”at 0.2 mM Rat [44] Orn,mMaKG 21 KPP mM aKG at 0.2 mM Rat Gabaculine Irreversible 0,020 mM [43] 0.5 aKGU OAT, “almost (liver) 2-aminooxyacetateAn intriguing property of 5-FMOrn is that even at highPLP,0.5 37concentrations, U °C, 60 OAT, mM40 complete” a small RatOAT activ[44]ity An intriguing property of 5-FMOrn is that even at highOrn, 37mMconcentrations, °C, 21 KPP 40mM at 0.2 mMa small OAT activity remains detectable in some tissues (brain and liver), whereas in other tissues inhibitionRat is complete An4-Aminohexynoate intriguing property of 5-FMOrnIrreversible is that even0,020mMaKG KPP mM at high concentrations, [43] a small OAT activity remains[20]. This detec activitytable is in associated some tissues with (brain kinetic and properties liver), whereas that are insomewhat other tissues different inhibition from(liver)Rat “standardis complete”; Gabaculine Irreversible PLP,0,020 60 mM mM [43] remains[20]. This detectable activity is in associated some tissues with kinetic (brain properties and liver), that are whereas somewhat indifferent“almost other from tissues(liver) “standard inhibition”; is complete [20]. thereAn is intriguingyet no satisfactory property explanationof 5-FMOrn foris thatthis evenfinding. at high OnePLP,Orn, concentrations,possibility 6021 mM could a smallbe the OAT weak activ OATity 2-aminooxyacetate complete” Rat [44] Thisthereremainsactivity activity is ofyetdetec another isnotable associatedsatisfactory intransaminase some tissuesexplanation with: GABA (brain kinetic for transaminaseand th liver),is propertiesfinding. whereas (EC One 2.6.1.19)Orn, in possibilitythataKG other21 mM is aretissues a potential atcould somewhat0.2 inhibition mM be candidate the isweak complete different, beOATing from “standard”; thereactivity[20]structurally is. This yet of activity noanother very satisfactory closeis transaminaseassociated to OAT andwith explanation: GABA present kinetic transaminase inproperties both for brain thisthat (EC and are finding.2.6.1.19) liver0.5 somewhat aKGU OAT,[34] is. a One differentpotential possibility fromcandidate “standard, couldbeing”; be the weak OAT structurallythere is yet veryno satisfactory close to OAT explanation and present for in th bothis finding. brain and One liver37 possibility °C, [34] 40 . “almostcould be the weak OAT activity2-aminooxyacetateAn of anotherintriguing property transaminase: of 5-FMOrn GABA is that even transaminase at high mMconcentrations, KPP (EC complete” 2.6.1.19) a small isRatOAT a potentialactiv[44]ity candidate, being activity of another transaminase: GABA transaminase (EC 2.6.1.19) is a potential candidateRat , being remainsGabaculine detectable in some tissues (brain and liver),Irreversible whereas 0,020in other mM tissuesat 0.2 inhibitionmM is complete[43] structurally very close to OAT and present in both brain and liver [34(liver)]. structurally[20]. This activity very close is associated to OAT andwith present kinetic inproperties both brain that and are PLP,liver somewhat 60 [34] mM. different from “standard”; Orn, 21 mM there is yet no satisfactory explanation for this finding. One possibility could be the weak OAT 2. StructureAn intriguing of OAT property of 5-FMOrn is that even at high concentrations,aKG a small OAT activity activity of another transaminase: GABA transaminase (EC 2.6.1.19) is a potential candidate, being remains detectable in some tissues (brain and liver), whereas in other tissues“almost inhibition is complete [20]structurally2-.aminooxyacetate This activity very closeis associated to OAT withand present kinetic inproperties both brain that and are liver somewhat [34]. differentcomplete” fromRat “standard [44]” ; 2.1. Primary Sequence and Leading Peptide at 0.2 mM there is yet no satisfactory explanation for this finding. One possibility could be the weak OAT activityThe human of another OAT transaminase (hOAT): GABA sequence transaminase is available (EC 2.6.1.19) on Uniprot is a potential (code candidate P04181,, being OAT_HUMAN), along structurallyAn intriguing very close property to OAT of and 5-FMOrn present is in that both even brain at and high liver concentrations, [34]. a small OAT activity withremains other detec OATtable or in putative some tissues OAT (brain sequences and liver), whereas (n = 53). in other The tissues “canonical inhibition sequence” is complete of the human OAT [20]. This activity is associated with kinetic properties that are somewhat different from “standard”; (Uniprot P04181-1) has a length of 439 AA and a molecular mass of 48,535 Da. A second isomorph is reportedthere inis yet the no database satisfactory (Uniprot explanation P04181-2) for this finding. with One the possibility first 138 could residues be the missing. weak OAT There has been a long activity of another transaminase: GABA transaminase (EC 2.6.1.19) is a potential candidate, being controversystructurally about very close differences to OAT and between present inliver both brain and and kidney liver [34] OAT,. some studies suggesting the existence of different isozymes [45,46] and distinct physicochemical properties [41]. However, other researchers have found no evidence for any difference between the liver, kidney and small intestine forms of rat OAT [18,47]. In organisms in which OAT is mitochondrial, the translated protein includes a signal peptide that targets the peptide to mitochondria. In humans, mutations in this peptide are one of the causes of gyrate atrophy. There is controversy about the length of the leading mitochondrial signal sequence. On UniProt, the cleavage location is reported to change between the kidney (after Q35, based on the work of Biology 2017, 6, 18 9 of 38

Kobayashi et al. [48]—unless otherwise specified, residue numbering is for human OAT) and the liver (after A25, based on the work of Simmaco et al. [49]) forms. However, a careful reading of the cited papers is inconclusive; Kobayashi et al. [48] suggest a similar cleavage for the kidney form, and mention a third possible cleavage site, after L32. Cleavage after Q35 was also described by Oyama et al. [50], who found exactly the same sequence between the renal and the liver OAT starting from this residue. A study on human retinoblastoma found a leading sequence of 32 residues, resulting in a mature protein of 407 residues [51]. Another study on rat liver concluded a leader sequence of 34 residues [52]. However, this was contested one year later, and a leader sequence of 25 residues was proposed [49]. A two-step cleavage of the signal peptide may explain why different cleavage sites were found. The sequence is positive for the Prosite [53] pattern PS00600, characteristic of the aminotransferases class-III PLP attachment site [54]. The consensus pattern is [LIVMFYWCS]-[LIVMFYWCAH]-x-D-[ED]-[IVA]-x(2,3)-[GAT]-[LIVMFAGCYN]-x(0,1)-[RSACLIH]- x-[GSADEHRM]-x(10,16)-[DH]-[LIVMFCAG]-[LIVMFYSTAR]-x(2)-[GSA]-K-x(2,3)-[GSTADNV]-[GSAC], where the conserved lysine residue is the pyridoxal-P attachment site. Among the 1070 UniProtKB/Swiss-Prot sequences having this pattern, 1031 are true positives. A multiple alignment of mammalian OAT sequences from the Uniprot database yields very high identities. The phylogenetic tree (Figure5) computed from this alignment is consistent with the close relatedness of all primates (100% identity of human OAT with the common chimpanzee and above 98% for other primates), but also near 90% identity with most mammals (rodents, canids, ruminants or bats), with the exception of elephant sequences, which have 75% identity with the human sequence. A close-up of the consensus Prosite pattern (Figure6) clearly shows the high degree of conservation of this key region (lysine residue PLP attachment site is in column 375 of the alignment, corresponding to residue 292 inBiology the 2017 human, 6, 1 OAT sequence). 12 of 40

Figure 5. FigurePhylogenetic 5. Phylogenetic tree tree computed computed from from multiple multiple alignment alignment distances of distances mammalian OATof mammalian sequences. OAT sequences.

Biology 2017, 6, 18 10 of 38 Biology 2017, 6, 1 13 of 40

Figure 6. Close-up of the Prosite consensus pattern region of the Clustal multiple alignment of mammalianFigure 6. Close OAT-up sequences. of the Prosite The pyridoxal consensus 5-phosphate pattern region (PLP) attachmentof the Clustal site multipleis the conserved alignment lysine of residuemammalian in column OAT 375 sequences. of the alignment The pyridoxal (denoted 5-phosphate by an asterisk), (PLP) which attachment corresponds site is to the residue conserved 292 in thelysine human residue sequence. in column 375 of the alignment (denoted by an asterisk), which corresponds to residue 292 in the human sequence. 2.2. The Three-Dimensional Structure 2.2. The Three-Dimensional Structure OAT belongs to the broad fold-type I structural group of PLP-dependent enzymes, and inside that groupOAT to the belongs class-III toaminotransferases the broad fold-type according I structural to Grishin group etof al.’s PLP nomenclature-dependent enzymes, [54] (corresponding and inside tothat the group group initiallyto the namedclass-III AT-II aminotransferases by Mehta et al. [ 55according]). Most of to these Grishin enzymes et areal.’sω -aminotransferases,nomenclature [54] with(correspond specificing features to the explained group initially by a number named of AT conserved-II by Mehta residues et al in. [55] the). active Most siteof th [56ese,57 enzymes]. Six crystal are structuresω-aminotransferases, of the human with OAT specific enzyme features arepresent explained in theby PDBa number databank: of conserved one of the residues recombinant in the enzymesactive site [[56,57]12], and. Six three crystal enzyme structures complexes of the human with 5FMOrn OAT enzyme [58], areL-canaline present andin the gabaguline PDB databank: [59], one of the recombinant enzymes [12], and three enzyme complexes with 5FMOrn [58], L-canaline and gabaguline [59], rerspectively, and two forms mutated on one or three residues [60]. All six

Biology 2017, 6, 18 11 of 38 rerspectively, and two forms mutated on one or three residues [60]. All six structures are isomorphous, and overall structures are almost identical, with only small differences on two flexible surface loops between free and complexed forms or on the mutated residues between wild and mutated ones. Each OAT monomer contains 12 α-helices and 14 β-strands, and forms a large and a small domain. The biologically active form is a dimer formed through extensive hydrophobic and hydrophilic interactions, essentially between the large domains of two monomers, burying approximately 5500 Å2 of solvent-accessible surface. The two active sites of the dimeric unit are 25 Å apart. The crystal packing creates a hexamer formed by a trimer of dimers related by an approximately 3-fold screw axis. Although it is not possible to tell from the crystal structure whether the hexamer is of biological relevance or merely an effect of crystal packing, there is experimental evidence for the existence of hexamers of rat liver OAT in solution approaching crystallization conditions [61]. Assemblies of dimers or tetramers have been observed in electron microscopic studies on pig kidney OAT [62], and oligomers are detected in high concentrations of mouse liver OAT [63]. The oligomerization state seems to change the kinetic properties of the enzyme [30], and may partly explain the differences observed between tissues, although the kinetic studies performed up till now have never shown any sign of cooperation between the various subunits (the kinetics seems to be of the Michaelis Menton type, see above).

2.3. The The PLP cofactor is fixed at the interface between subunits in the large domain, forming an internal aldimine via a Schiff base bond with K292. Crystal structures of OAT complexed with inhibitors have been studied in order to elucidate the exact mechanism of the reaction. The suicide inhibitor 5FMOrn is an Orn analogue irreversibly binding in the OAT active site in the initial transamination step [58]. 5FMOrn forms a stable adduct with the PLP cofactor, referred to as FMP, which is detached from the K292 residue, but is kept firmly in the active site through non-covalent interactions with OAT residues (Figure7). This crystal structure suggests a mechanism for the specific ω-transaminase function of OAT [58]: the α-amino and α-carboxy groups of Orn are anchored to Y55 and R180 respectively (mutations Y55S and R180T on these two residues cause loss of OAT activity, and lead to gyrate atrophy [64,65]); thus only the δ-amino group can approach the reactive Schiff adduct of K292-PLP. Arginine R413 could bind the ornithine α-carboxylate group instead of R180: This arginine (R413) is one of the four residues conserved in all transaminases [55]; in other transaminases, it binds the α-carboxylate group of the substrate through a hydrogen bond [66,67]. Conversely, in OAT, residue E235 forms a tight ion pair with R413, making it unavailable for hydrogen bonding. It is suggested that this shielding leaves no alternative binding residue for the α-carboxylate other than the R180, which accounts for why OAT transaminates the ω instead of the more reactive α-amino group in its first half-reaction. However, this hypothesis was challenged by a crystal structure study of an OAT with glutamate 235 mutated to alanine [60]. This mutation conserved specific transamination of the δ- instead of the α-amino group of ornithine, while at the same time shifting the specificity from ornithine to glutamate. An E235S mutation produced similar qualitative results, but on a lesser scale. In addition, the interaction between residues E235 and R413 is stabilized via a hydrogen bond network involving the PLP form of the cofactor, which is weakened when the cofactor is in its PMP form. The authors [57] suggest that E235 acts as a switch, closed in the E-PLP form, thereby allowing binding of ornithine for ω-transamination, but impeding binding of glutamate, and opened in the E-PMP form, making R413 available to bind aKG. We note that this model is consistent with the experimental evidence that, in vitro, OAT more easily catalyzes Orn degradation. BiologyBiology2017 2017, ,6 6,, 18 1 1215 of of 38 40

Figure 7. Active site geometry. Image created with JMol using the OAT-5FMOrn crystal structure Figure 7. Active site geometry. Image created with JMol using the OAT-5FMOrn crystal structure coordinates. The ball and stick structure shows the adduct formed by the reaction of 5FMOrn with PLP. coordinates. The ball and stick structure shows the adduct formed by the reaction of 5FMOrn with The wireframe structures indicate the residues stabilizing the adduct in the active site in particular Y55, PLP. The wireframe structures indicate the residues stabilizing the adduct in the active site in R180, K292, and the shielding of R413 by E235. particular Y55, R180, K292, and the shielding of R413 by E235. 3. Synthesis and Fate of OAT—From Gene to Protein 3. Synthesis and Fate of OAT—From Gene to Protein 3.1. From Gene to Protein 3.1. From Gene to Protein 3.1.1. Genes Encoding OAT 3.1.1. Genes Encoding OAT In humans, OAT is coded by a single gene, on 10, region q26 [68,69] (positions 124397303In human to 124418976s, OAT is incoded human by genomea single gene, 19; also on namedchromosome OKT, 10, GACR, region OATASE q26 [68,69] and (positions HOGA). In124397303 addition, to there124418976 are at in least human three genome pseudogenes 19; also ofnamed very closeOKT, sequenceGACR, OATASE similarity: and twoHOGA). on the In X-chromosomeaddition, there [69are] atat Xp11.2 least three [68] (OATL1) pseudogenes and Xof (OATL2), very close and sequence one on chromosome similarity: two 10, nexton the to theX-chromosome original gene [69] (OATL3). at Xp11.2 Proof [68] of (OATL1) the functionality and X (OATL2 of the), gene and onone chromosome on was 10, established next to the byoriginal transfection gene (OATL3). and expression Proof of in hamsterthe functionality ovarian cellsof the [70 gene]. The on pattern chromosome is similar 10 inwas the established mouse, with by atransfection gene on chromosome and expression 7 and in pseudo-geneshamster ovarian on cells [70]. The pattern X and is 3 similar [71]. in the mouse, with a gene on chromoInterestingly,some 7 and the pseudo OAT- genegenes promoteron chromosomes region X sharesand 3 [71] properties. of low-level, ubiquitous house-keepingInterestingly, genes the (rich OAT GC content,gene promoter Sp1 binding region sites) share ands bindingproperties sites of for low several-level, transcription ubiquitous factorshouse-keeping found for genes more (rich tissue-specific GC content, genes. Sp1 binding This finding sites) and is consistent withs for the several low levels transcription of OAT foundfactors in found almost for all more tissues, tissue and-specific higher genes. expression This finding in a few is consistent tissues. with the low levels of OAT found in almostLittle all is knowntissues, aboutand higher regulation expression by miRNAs, in a few with tissues. only one mention in the literature of a putative OAT geneLittle predictedis known about as the regulation target of fourby miRNAs, different with miRNAs only one in Amphioxus mention in[ 72the]. literature In humans, of a the putative OAT transcriptOAT gene is predicted one target as ofthe hsa-miR-1 target of four [73,74 different], which miRNAs is involved in Amphioxus in muscle [72] and. In heart humans, development. the OAT Thistranscript finding is isone consistent target of withhsa-miR the-1 recent [73,74] discovery, which is of involved the importance in muscle of and OAT heart in muscles, development. and of This its finding is consistent with the recent discovery of the importance of OAT in muscles, and of its changes

Biology 2017, 6, 18 13 of 38 changes during growth [75]. Additionally, the possibility arises of a link between the existence of OAT pseudo-genes on the X chromosome, the fact that pseudo-gene transcripts were recently discovered to act as a trap for miRNAs, and the higher expression of OAT in females. Two teams [76,77] claimed a link between sarcoma cell lines and mutations in the X chromosome near the OATL1 and OATL2 loci. A few other hsa-miRs are also predicted in silico to interact with OAT transcript [78], but with no experimental evidence to date.

3.1.2. Transcription Transcription occurs on the (−) strand, and there seem to be several variants of the transcript. The primary transcript of the OAT gene is 21 kb long and contains 11 . 2 is, however, spliced out [79], except in some retinoblastoma cell lines, where a few mRNAs escape this splicing [80]. Except for this particular case, there is no experimental evidence for regulation through alternative splicing. Transcription control by glucagon and glucocorticoids of the cycle enzymes, as occurs in the liver, needs a CCAAT regulation site and the fixation of the CCAAT enhancer-binding protein β [81]. Since the OAT gene promoter also has CCAAT regions, and glucagon also increased OAT activity in the liver, a similar mechanism is probably in play.

3.1.3. Translation Translation starts in exon 3, and seems to occur on free polysomes [82]. It involves the eIF-4E translation factor [29], except for the few mRNAs that do not splice out the exon 2, which are less translated, and independently of the eIF-4E level. The reason for this is not clear, two possible explanations being the fixation of a protein (or of miRNA) and the longer prefix sequence, facilitating the formation of mRNA secondary structures inhibiting the binding of translation factors. Protein-protein interaction studies have also identified eIF-6 as interacting with OAT [83], suggesting it may also be involved.

3.2. Changes in Protein

3.2.1. Peptide Maturation Since OAT is mitochondrial in most species, the translated peptide must be translocated into the mitochondrial matrix. This is done using a signal peptide that is removed after translocation. Orientation toward the mitochondrion first occurs by the binding of the precursor to a receptor on the mitochondrial outer membrane; the signal peptide is removed after crossing this membrane [84]. As dealt with in detail in 2.1, there is some controversy about the exact length of this signal peptide.

3.2.2. Post-Translational Modifications Three kinds of post-translational modifications are experimentally proven for mammalian OAT and are summarized in Figure8. The first one is the fixation of the PLP cofactor in the active site, already described in detail (2.3). The second one was detected in mice, and corresponds to N-acetylation and N-succinylation of lysine residues. Seven Lys residues of OAT can be succinylated in mouse liver [85] in positions 102, 107, 362, 374, 386, 392 and 405, and desuccinylation occurs through Sirt-5, a sirtuin enzyme. Conversely, 8 Lys residues can be acetylated [86] in positions 49, 66, 107, 362, 386, 392, 405 and 421, and deacetylation occurs through Sirt-3. We note that five of these positions are common to succinylation sites. The exact consequences of these post-translational modifications are not known. They may be involved in the differences in OAT kinetics noted among various organs (see Section1). Biology 2017, 6, 18 14 of 38 Biology 2017, 6, 1 17 of 40

Figure 8. Amino acid sequence alignment of human, rat, mouse and ox OAT. Figure 8. Amino acid sequence alignment of human, rat, mouse and ox OAT.

ThereThere seemsseems to to have have been be noen experimentalno experimental mention mention to date to of date any otherof any post-translational other post-translational changes, includingchanges, including a potential a enzymepotential regulatory enzyme regul modificationatory modification (e.g., nitrosylation, (e.g., nitrosylation, phosphorylation). phosphorylation However,). large-scaleHowever, experimentslarge-scale experiments and computer and predictions computer suggest prediction that sumoylations suggest [that87,88 ],sumoylation neddylation [87,88] [88,89], andneddylation ubiquitination [88,89] [and90–94 ubiquitination] can occur, and [90 that–94] therecan occur, may beand several that there kinase may target be several sequences. kinase target sequences. 3.3. Life and Death of the Protein

3.3.1.3.3. Life Interaction and Death with of the Other Protein Proteins

3.3.1.Several Interaction protein-protein with Other Proteins interactions involving OAT have been predicted in silico or found in large-scale studies, prompting several hypotheses on the functions of OAT and on its regulation. Several protein-protein interactions involving OAT have been predicted in silico or found in Proteins involved, detected in a very large-scale co-precipitation study [95], with some of the large-scale studies, prompting several hypotheses on the functions of OAT and on its regulation. interactions that were only predicted, are NME2 (nucleotide diphosphate kinase, involved in tumorigenesis),Proteins involved, EZR detected (ezrin), in the a aspartatevery large aminotransferase-scale co-precipitation GOT1 study (or more [95] probably, with some GOT2, of the its interactions that were only predicted, are NME2 (nucleotide diphosphate kinase, involved in tumorigenesis), EZR (ezrin), the aspartate aminotransferase GOT1 (or more probably GOT2, its

Biology 2017, 6, 18 15 of 38 mitochondrial equivalent); ESD (esterase D, a marker of retinoblastoma), AKR1B1 (reducing aldehydes and ketones), ALDH1B1, CBS, SOD1 and SOD2, PDXK (pyridoxal kinase), UAP1L1, UQCRFS1, PREP, PGM1, PITPNB, PPID and ADSS, the biological relevance of which is still difficult to assess. Other studies have detected interactions with NTRK1 [96], HDAC5 (a histone deacetylase [97]), FAF2 [98], NUDT3 [99], SIRT-7 [100], ICT1 [101], SLC35F6 [102], OTUD4 [103], DMWD [103], CDK2 [104], UBQLN4 [105], HUWE1 [106], FUS1 [107], EGFR [108], ADRB2 [109], FBXO6 [110], CTSA [111], ATF2 [112]. However, to our knowledge, there is no experimental confirmation that these interactions occur in normal cells under physiological conditions. For instance, interaction between ATF2 and OAT was detected in an experiment in which it was concluded that ATF2 targeted the mitochondrial outer membrane through interactions with its proteins, but since OAT is in the mitochondrial matrix, the reason for the detection of ATF2-OAT pairs is not clear. A special case is the interaction with c-MYC, a transcription factor involved in cell proliferation: protein-protein interaction was detected in a large-scale study [113], but conversely, c-MYC was found to increase the OAT mRNA levels, and other enzymes involved in Glu metabolism, in activated T-cells [114], which use Glu as a precursor for Orn, which in turn is a precursor of polyamines. Hence this OAT-cMYC interaction might be a negative feedback, but no experimental evidence has yet provided any insight into the precise role of this interaction.

3.3.2. OAT Degradation The OAT degradation rate constant is around 0.4 day−1 in the liver [35,115,116], i.e. a half-life of about 1.7 days (but only 0.95 days in the rat [117]). The degradation rate seems to be much slower in the kidney, with a half-life of about 4 days in the rat [117]. It may start by the loss of its PLP cofactor [118]. Since sumoylation, neddylation and ubiquitination are usually involved in protein degradation control, the occurrence of these various binding sites may explain differences in OAT half-lives according to the different organs.

4. Localization of OAT

4.1. Cellular Localization In all known species with OAT activity, OAT is a soluble, intracellular protein. In eukaryotic cells, the cellular localization of OAT varies among different phyla. It is a cytosolic enzyme in fungi (shown for Neurospora [119] and Saccharomyces cerevisiae [120], and suspected for other fungi (Agaricus bisporus [51,121], Saccharomycetae [26,121,122])) and in most unicellular organisms, but it is mitochondrial in most plants and vertebrates. In all mammals investigated thus far OAT is in the mitochondrial matrix. This localization is consistent with the fate and origin of the metabolites it uses, since most pathways with which OAT interacts involve mitochondrial enzymes (Figure1). This mitochondrial localization may also control the reaction kinetics, OAT activity being apparently limited by the rate of ornithine entry into the mitochondrion [22]. There are also consistent reports that OAT is present in the cell nucleus, where its function is still unknown. Several observations suggest it could be an actor in cell division and function. First, inhibition by diazonamide A, a natural toxin with anti-mitotic properties, was shown to act through OAT in cancer cell lines [123], but without inhibiting its activity. Surprisingly, this interaction does not seem to block normal cell division, but only tumoral cell division. Second, a recent investigation of the changes in the Staufen-1 complex induced by HIV identified OAT as one of the numerous proteins involved in this complex, which binds mRNAs and targets them to different cellular organelles (of note, OAT mRNA was not identified as one of the mRNAs bound to various Staufen complexes) [124]. This observation may be related to the finding that mutations in mitochondrial enzymes, such as OAT, influence HIV-1 propagation [125]. Third, bioinformatics analysis of the OAT gene sequence predicts the existence of binding sites for several transcription factors involved in cell cycle or cancer genesis, such as EVI-1 (two sites). A direct effect of the c-MYC transcription factor was also demonstrated [114]. However, to our knowledge, no experimental evidence for the binding of these transcription factors to the OAT gene has been reported in the literature. Hence future investigation of the nuclear function of OAT may reveal unexpected functions. Biology 2017, 6, 1 19 of 40 binding of these transcription factors to the OAT gene has been reported in the literature. Hence

Biologyfuture2017 investigation, 6, 18 of the nuclear function of OAT may reveal unexpected functions. 16 of 38

4.2. Tissue Localization 4.2. Tissue Localization Before summarizing the information about OAT location in mammalian organs and tissues, someBefore discussion summarizing of the experimental the information methods about OATused locationare needed, in mammalian to reconcile organs apparently and tissues, conflicting some discussionresults. Historically, of the experimental OAT has been methods first detected used are in needed, organs to and reconcile tissues apparently through its conflicting catalytic activity, results. Historically,monitored in OAT general has been by firstthe detecteddetection in organsof 14C- andradiola tissuesbeled through Orn degradation its catalytic activity, products, monitored either inglutamate general byor theP5C, detection or by chemical of 14C-radiolabeled reactions of Orn the degradation product. This products, has been either done glutamate on enzyme or P5C,s of orvarying by chemical purity, reactionsorgan extracts of the or product. even in This vivo. has These been experiments, done on enzymes especially of varying if the purity,enzyme organ was extractspurified, or yield even conclusivein vivo. These evidence experiments, that the enzyme especially occurs if the in enzymean active was form purified, in the organ yield considered conclusive. evidenceHowever, that it give the enzymes no insights occurs ininto an its active exact form localization in the organ in considered. the tissue, However,and seldom it gives into no the insights exact intoreaction its exact taking localization place physiologically, in the tissue, andbecause seldom the into reaction the exact direction reaction is takingdetermined place physiologically,by the relative becauseconcentrations the reaction of substrates direction and is determinedproduct. by the relative concentrations of substrates and product. Later, directdirect detectiondetection ofof thethe proteinprotein waswas achievedachieved usingusing specificspecific antibodies.antibodies. Coupled with fluorescence,fluorescence, this allows the precise detection of the protein in each cell, thereby giving precise informationinformation onon thethe histological histological location location of of OAT. OAT. However, However, failure failure to detect to detect the protein the protein may alsomay mean also thatmean it isthat in ait different is in a conformationaldifferent conformational state or has state undergone or has differentundergone post-translational different post modifications,-translational notmodifications, recognized not by therecognized antibody. by It the may antibody. also result It frommay thealso antibody result from binding the antibody site being binding masked site by anbeing interaction masked withby an another interaction protein. with In addition,another protein. it tells us In nothing addition, about it tells the activityus nothing of the about enzyme. the Typicalactivity histologicalof the enzyme. views Typical of OAT histological localization views can of be OAT found localization in the Human can be Protein foundAtlas in the [ 126Human,127], obtainedProtein Atlas using [126,127] two different, obtained antibodies using two (Figure different9). antibodies (Figure 9).

Figure 9. Protein expression scores from the Human Protein Atlas. Figure 9. Protein expression scores from the Human Protein Atlas.

The most recent detection method is the semi-quantitative detection of OAT mRNA, as can be foundThe in mostseveral recent gene detection expression method databases, is the such semi-quantitative as the Human detection Protein Atlas of OAT or mRNA,Bgee [128,129] as can be(although found inolder several experiments also quantified databases, mRNA such asinthe some Human circumstances Protein Atlas and ordevelopment Bgee [128,129 of] (althoughmicro-arrays older greatly experiments scaled up also these quantified data). mRNAHowever, in somethere circumstancesis not necessarily and developmentany correlation of micro-arraysbetween the amount greatly scaledof mRNA up these and data).the protein However, content there or is its not enzymatic necessarily activity. any correlation Expression between levels the amount of mRNA and the protein content or its enzymatic activity. Expression levels based on based on microarray data are available [130,131] (Figure 10). microarray data are available [130,131] (Figure 10). OAT can be found in almost all tissues (see Table3), but its activity predominates in the liver, kidney, intestine, and retina. As regards protein concentration or mRNA content, the intestine is clearly the organ in which OAT is most abundant, with both high mRNA and high protein content, whereas other tissues have moderate protein content and low RNA levels [Protein Atlas.org]. Discrepancies between activity and protein content measured by immunochemistry may result from different aggregation of the protein when its concentration changes (see 2.2), with effects on both its activity [30] and the antibody fixation.

Biology 2017, 6, 18 17 of 38 Biology 2017, 6, 1 20 of 40

Figure 10 10.. ProteinProtein expression levels from microarray data.

OAT can beTable found 3. OAT in almost activity all measured tissues in(see various Table tissues, 3), but with its activi no enzymety predomina isolation. tes in the liver, kidney, intestine, and retina. As regards protein concentration or mRNA content, the intestine is clearlyOrgan the organ in which Tissue OAT is most abundant Species, with both Activity high mRNA and Unithigh protein Referencecontent, Eye Neuroretina Cat 88.2 mmol/kg dry wt/h [70] whereas other tissues have moderate proteinOx content and 71 ±low17 RNAnmol/mg levels protein/30 [Protein min Atlas.org]. [132] Optic nerve Cat 6.7 mmol/kg dry wt/h [70] Discrepancies betweenTapetum activity and protein content Cat measured26.7 by immunochemistrymmol/kg dry wt/h may result [70 from] Sclera Cat 5.36 mmol/kg dry wt/h [70] different aggregation Choroidof the protein when its concentrationCat changes 17.4 (see 2.2),mmol/kg with dry effects wt/h on both [70] its Ox 14 ± 5 nmol/mg protein/30 min [132] activity [30] and the antibodyRetina fixation. Rat 174 ± 25 nmol/mg protein/h [133] 97 ± 9 nmol/mg protein/30 min [132] Ox 119 ± 34 nmol/mg protein/h [133] Table 3. OAT activity measured in variousPig tissues, 409 with± 50 no enzymenmol/mg isolation. protein/h [133] Dog 159 nmol/mg protein/h [133] Chicken 124 ± 16 nmol/mg protein/h [133] Organ Tissue Species Activity120 ± 20 Unitnmol/mg/h Reference [134] Eye Neuroretina CatTurkey 88.2 85 mmol/kgnmol/mg dry wt/h protein/h [70] [133 ] Rana pipiens 302 nmol/mg protein/h [133] RanaOx clamitans 71 ± 17 238 nmol/mg protein/30nmol/mg protein/hmin [132] [133 ] Xenopus laevis 392 ± 93 nmol/mg protein/h [133] Optic nerve CyprinusCat carpio 6.7 766 ± 57 mmol/kgnmol/mg dry wt/h protein/h [70] [133 ] Pigment epitheliumTapetum (iris epithelium) Cat Cat26.7 138 mmol/kgmmol/kg dry wt/h dry wt/h[70] [70 ] Rat 988 ± 130 nmol/mg protein/h [133] Sclera Cat Ox5.36 497 ± 89 mmol/kgnmol/mg dry wt/h protein/h [70] [133 ] Choroid Cat 17.4 113 ± 24 mmol/kgnmol/mg dry wt/h protein/30 min[70] [132 ] Pig 2260 ± 630 nmol/mg protein/h [133] Ox Dog14 ± 5 602 ± 159nmol/mg protein/30nmol/mg protein/hmin [132] [133 ] Retina RatChicken 174 ± 143125 ± 535 nmol/mgnmol/mg protein/ protein/hh [133] [133 ] 1670 ± 55 nmol/mg/h [134] Turkey 97 ± 9 1150 nmol/mg protein/30nmol/mg protein/hmin [132] [133 ] Rana pipiens 795 nmol/mg protein/h [133] RanaOx clamitans 119 ± 34 217 nmol/mgnmol/mg protein/ protein/hh [133] [133 ] XenopusPig laevis 409 ± 50171 ± 87 nmol/mgnmol/mg protein/ protein/hh [133] [133 ] Cyprinus carpio 203 ± 51 nmol/mg protein/h [133] Vascular layer of the ciliary body Dog Cat159 63.0 nmol/mgmmol/kg protein/ dryh wt/h[133] [70 ] Cornea Chicken Cat 124 ± 16 8.11 nmol/mgmmol/kg protein/ dryh wt/h[133] [70 ] Ox 14 ± 9 nmol/mg protein/30 min [132] Lens Cat120 ± 20 No nmol/mg/h [134][70 ] TurkeyOx 85 No nmol/mg protein/h [133][132 ] Brain Frontal cortex Rat 1.45 ± 0.29 mU/mg protein [135] Occipital cortex Rana pipiensRat 3021.38 ± 0.08 nmol/mg proteinmU/mg/ proteinh [133] [135 ] Olfactory bulb Rat 1.23 ± 0.07 mU/mg protein [135] Hippocampus Rana clamitans Rat 2381.33 ± 0.16 nmol/mg proteinmU/mg/ proteinh [133] [135 ] Striatum Xenopus laevis Rat 392 ± 0.9593 ± 0.06 nmol/mg proteinmU/mg/ proteinh [133] [135 ] Midbrain, thalamus Rat 1.17 ± 0.12 mU/mg protein [135] Hypothalamus Cyprinus carpioRat 766 ± 1.3457 ± 0.10 nmol/mg proteinmU/mg/ proteinh [133] [135 ] Pigment epitheliumPons, medulla (iris oblongata epithelium) Cat Rat1380.85 ± 0.04 mmol/kg dmU/mgry wt/h protein [70] [135 ] Cerebellum Rat 0.90 ± 0.19 mU/mg protein [135] Tractus opticusRat Rat988 ± 130 0.75 ± 0.10 nmol/mg proteinmU/mg/ proteinh [133] [135 ] Spinal cord Ox Rat497 ± 0.6089 ± 0.07 nmol/mg proteinmU/mg/ proteinh [133] [135 ] 113 ± 24 nmol/mg protein/30 min [132] Pig 2260 ± 630 nmol/mg protein/h [133] Dog 602 ± 159 nmol/mg protein/h [133] Chicken 1431 ± 535 nmol/mg protein/h [133] 1670 ± 55 nmol/mg/h [134] Turkey 1150 nmol/mg protein/h [133]

Biology 2017, 6, 18 18 of 38

Table 3. Cont.

Organ Tissue Species Activity Unit Reference Liver Rat 179 nmol/mg protein/h [133] 242 ± 41 nmol/mg protein/30 min [132] Pig 164 nmol/mg protein/h [133] Ox 12 nmol/mg protein/30 min [132] Chicken 174 ± 22 nmol/mg protein/h [133] 150 ± 40 nmol/mg/h [134] Rana clamitans 137 nmol/mg protein/h [133] Xenopus laevis 161 ± 101 nmol/mg protein/h [133] Cyprinus carpio 590 nmol/mg protein/h [133]

4.3. OAT in the Liver As noted above, the liver is one of the organs with the highest OAT activity, and one of those most widely studied in the mouse, rat and pig, and in humans. Interestingly, despite this high activity, RNA levels and protein levels detected by immunohistochemistry are quite low, except in the gallbladder [136]. Beside the explanations given above, another possibility is the change in cell conditions, liver OAT activity depending on the protein content of the diet. Whereas early work suggested OAT involvement in ornithine synthesis [46,137–139] to modulate its availability for the urea cycle, further experiments suggested that OAT instead removed excess ornithine to prevent an increase in urea cycle metabolites [22]. Involvement in gluconeogenesis has also been proposed [140]. However, most of these experiments were conducted using all types of hepatocytes (i.e., periportal and perivenous), complete liver, or even in vivo: thus, the results must be interpreted with caution because expression experiments suggest that OAT and the urea cycle enzymes are expressed in distinct cells [141], with OAT expressed in pericentral hepatocytes in the adult mouse [142] and rat [143]. This is in line with histochemical experiments carried out on rats [144], which found an increasing gradient of OAT from the periphery to the center of the lobule. Colocalization of OAT and (Gln) synthase in these cells suggests that OAT might be used to generate Glu: With the rapid reduction of GSA to Glu in the mitochondria [22], each ornithine molecule allows the generation of two Glu through this pathway [139]. Since aKG, as a Krebs cycle intermediate, should not be limiting, this seems to be a very efficient way to supply Gln [145]. Interpretation of OAT activity experiments in the liver, without considering cell type and localization in the lobule, may also be biased by a potentially higher expression of OAT in the gallbladder [136], and so putatively in its afferent canals. The role of OAT in the gallbladder is unknown, but the OAT gene promoter may contain an aryl hydrocarbon receptor (AhR) and an AhR nuclear translocator (ARNT) binding site [in silico prediction], and AhR is known to bind bilirubin; this opens a possible line of enquiry. In the liver, OAT activity increases after a high protein diet [146–148], an amino acid-enriched diet, or stimulation by glucagon [149,150] or cAMP [140]. Effect of amino acids is indirect and mediated by glucagon, which acts by increasing cAMP levels. However, this action occurs only in the presence of (or after a pre-treatment by) cortisone [150]. , fructose, L-sorbose, and glycerol seem to inhibit this regulation process [140,146,148,151], with an efficacy that varies between in vivo and cultured cells. In all situations, the effect seems to be on the translation step, with increased rate of synthesis [149], and little or no effect on transcription levels [152]. How hexoses and glycerol act is not yet fully understood. OAT activity is also increased by all-trans-retinoic acid (atRA) [153], through transcription enhancement. Conversely, OAT activity is decreased by glucocorticoids [154,155], and by lipopolysaccharide (LPS) [156]. In mice and rats, it presents circadian variations, with a decrease along the day, by control of the translation step, but with no change in the mRNA levels [17,149,157], and with an effect of the dark/light transitions [158]. Oxygen pressure also seems to have an influence, with low levels increasing OAT activity, which may be responsible for the higher activity of OAT in pericentral hepatocytes [143]. The mechanism is not completely elucidated, but may involve a heme protein and share the same mechanism as glucagon and cAMP. Another explanation could be the increased Biology 2017, 6, 18 19 of 38

+ NH4 concentration at low O2 pressure (owing to a slower urea cycle rate), lowering OAT degradation. A third, putative explanation based on in silico predicted features of the OAT gene is the presence of an ARNT binding site, ARNT being known to interact with HIF-1a to form hypoxia-induced factor 1 (HIF1). Since the experiments of Colombatto et al. [143] did not directly show the involvement of a heme protein, but only that cobalt chloride increased OAT activity (even though CoCl2 is a known direct inhibitor of OAT [23]), and since it is known that CoCl2 induces HIF-1 [159], this regulation pathway seems consistent, although it awaits confirmation.

4.4. OAT in the Digestive Tract In the intestine, OAT is believed to catalyze the formation of Orn, which is subsequently converted into Cit, and ultimately into Arg under conditions where argininosuccinate synthase (ASS) and argininosuccinate (ASL) are expressed (see below for details). The intestine is the main organ presenting this flux characteristic. The importance of this reaction changes during growth, as will be detailed in 6.1 (briefly, in neonates Pro is an Arg precursor, and lack of OAT has adverse effects; mice lacking OAT are not even viable without Arg supplementation in the first weeks); the focus here will be on adult mammals, in which OAT is used to produce Cit from Glu. Considering the equilibrium constant of the reaction, taking this direction requires either a large amount of GSA and Glu, or fast consumption of ornithine (or aKG). This is in line with its conversion into citrulline, which in turn is released in the portal circulation. Large amounts of Pro may also lead to ornithine synthesis by OAT [160]. In the small intestine, none of the activators at work in the liver or kidney seem to have any effect. It has been shown that a low protein diet induces intestinal OAT [161], and that all-trans-retinoic acid (atRA) induces an increase in OAT activity during the differentiation of villus cells [162].

4.5. OAT in the Kidney In the kidney, OAT converts ornithine and α-ketoglutarate into P5C and Glu, respectively. Interestingly, females have about twice as much OAT protein as males [18]. In mice, OAT activity seems to be higher in the proximal than in the distal tubule [163]. In the kidneys, OAT activity is insensitive to glucagon and high protein diet [164], but is increased by thyroid hormones [165] and estrogens [117,152,164,166]. By contrast, testosterone seems to decrease its activity [167]. This stimulation by estrogens and inhibition by testosterone doubtlessly explains the higher OAT activity in female kidneys. Tri-iodotyrosine (T3) may act through cAMP. Estrogens seem to act by accelerating the translation initiation, whereas T3 changes the transcription [168].

4.6. OAT in the Brain and the Nervous and Sensory System In the brain, OAT is found in cortical, hippocampal and basal ganglionic neurons [169] and, at a lower level, in glial cells [136]. It is thought to be used for Pro synthesis [135], but has also been proposed as a source of Glu, a precursor for different kinds of neurotransmitters (Glu itself [170], GABA [171–173]). This last idea is supported by the high amount of OAT found in nerve endings [174], and by a decrease in OAT activity in brain anoxia models, in tissues where GABAergic neuron degradation occurs. However, other experiments suggest that GABA is not derived from Orn [42,170]. GABA itself has an inhibitory effect on OAT that could be due to substrate competition with Orn (see above) [42]. This was observed in vivo by peritoneal administration of vigabatrin, leading to an increase in GABA levels and Orn levels, and confirms the Orn → GSA/Glu function of OAT in the brain. The link between OAT and Glu as a neurotransmitter has been mentioned to account for the lower levels of OAT found in the brains of patients who have died from Huntington’s chorea, compared with other dementias [175,176]. Another putative connection is the well-established fact that bilirubin is toxic for nerves, and that its toxicity is accompanied by increased Glu: since the OAT gene may have an AhR binding site (see above) and AhR binds bilirubin, exploring OAT expression and activity may help to elucidate the mechanism of bilirubin toxicity. Biology 2017, 6, 18 20 of 38

OAT is also found in several parts of the eye (Table1;[ 132,177,178]). Although its role is currently unknown, this finding is consistent with the ophthalmic consequences of OAT dysfunctions, as seen in gyrate atrophy (see 6.2). OAT activity in human retinoblastoma cells seems to be regulated as in kidney cells [80].

4.7. OAT in the Other Tissues In other organs and tissues, OAT is viewed as a housekeeping protein, controlling ornithine availability for the cell: microarray expression experiments have detected OAT mRNA in at least 131 different types of tissue [Bgee gene expression database] [129]. As Orn is a precursor for polyamines, OAT is involved in controlling the proliferation of several cell lines, such as colonocytes [179] and vascular smooth muscle cells [180]. There is also evidence that OAT is involved in activated T-cells, to provide both ornithine and aKG [114]. Conversely, controlling the metabolism of Orn into Pro makes OAT a modulator of collagen synthesis, and thus of extracellular matrix formation [180]. Attention has recently focused on OAT in muscles [181]. In terms of the protein atlas, the protein was found in heart and skeletal muscle cells, but not in smooth muscle cells, whereas RNA is found in all kinds of muscle cells. Often underestimated, the role of muscle OAT seems particularly important: although specific OAT activity per unit mass of protein in muscle is lower than in other tissues (intestine, liver and kidney), the much higher muscle mass in the body compared with these organs, implies a muscle contribution to whole body OAT activity similar to that of the liver in adult mice [75]. The exact role of OAT in muscle is unclear. The OAT regulation in the muscle seems similar to that in the kidney, with lower levels in males than in females, which may result from either estrogen stimulation in women or testosterone inhibition in men.

5. The Role of OAT in Ornithine Fluxes As noted above, the reaction catalyzed by OAT favors Orn degradation in vitro, but there is evidence that the equilibrium constant is low enough to allow Orn synthesis under certain circumstances in vivo. The reaction direction is controlled by relative metabolite concentrations, which is difficult to appraise without a general view of the pathways in which OAT is involved. Throughout the following discussion on Orn fluxes we must keep in mind the direction of flux to be considered: net Orn degradation by OAT at the whole body level does not preclude net Orn synthesis locally in some specific organs or in particular situations.

5.1. Fluxes in Non-Mammals: Orn Degradation For organisms other than mammals, the picture seems quite simple: all the available evidence suggests that OAT catalyzes Orn degradation into Glu and GSA/P5C. The end-product of ornithine by this pathway can be either Glu or Pro, and there is frequent controversy about which it is. This simplicity may be related to the existence of a completely separate pathway producing Arg, involving acetylated derivatives of amino acids.

5.1.1. Prokaryotes In prokaryotes, OAT (rocD gene) is involved in one of the Arg degradation pathways, allowing the formation of Pro, the first step being Arg deimination into Cit, which in turn is further converted into Orn. However, the full picture may be more complex, because the arginine pathway involves a closely related enzyme, N-acetylornithine δ-aminotransferase (EC 2.6.1.11; NAcOAT, argD gene), which can also act on ornithine. Furthermore, this degradation pathway is not the main one, and is used in conditions where no better source of nitrogen than Arg is available. Quite often only the first two steps take place, as an energy-producing pathway. Lastly, arginine catabolism can also proceed through succinylated compounds; an N-succinylornithine δ-aminotransferase (EC 2.6.1.81, SOAT) operates, is close to OAT, and may also act on ornithine. Hence proving the existence of a functional OAT, distinct from NAcOAT and SOAT, is difficult: its existence in Archaeobacteria is, for example, still debated, as to date it has been detected only in genome analysis. Even the existence of the gene in the roc operon is no proof, recent work having shown that Mycobacterium tuberculosis (and other species causing tuberculosis) has a non-functional rocD gene, unlike other Mycobacteria [182]. Biology 2017, 6, 18 21 of 38

5.1.2. Plants In plants, where the arginine biosynthesis pathway also uses N-acetylated precursors in the production of ornithine, the situation is similar to that in bacteria, and OAT is believed only to degrade ornithine. However, its role is a subject of debate and may depend on the organism. Some authors claim that OAT is involved in resistance against some kinds of stress, especially salt stress and drought stress, through the synthesis of Pro (which is regarded as a reactive oxygen species [ROS] acceptor and as an osmolyte) that accumulates in vacuoles. OAT activity is indeed increased by salt (NaCl) stress in radish seeds (Raphanus sativus)[121], probably through increased translation from already-formed mRNA [183], Arabidopsis thaliana seeds [122], and by drought and oxidative stress in rice (Oryza sativa), where OAT gene transcription is under the control of an SNAC2 gene that codes for a transcription factor involved in the stress response [184]. However, in other plants such as Vigna aconitifolia, this pathway is used when nitrogen is in excess [28], and other works suggest that OAT is used in the seeding process to enhance Gln production. Inclusion of the A. thaliana OAT gene in other plants induced an increased resistance to drought and salt stress that may be useful in their cultivation (e.g., tobacco, Nicotiana plumbaginifolia, or squash, Cucurbita pepo)[121].

5.1.3. Fungi In fungi, from which one of the first OATs was identified (in Neurospora crassa [185]), OAT seems to be involved in the synthesis of Pro [186](Aspergillus nidulans) and Glu.

5.1.4. Unicellular In unicellular eukaryotes, OAT also mainly acts as an ornithine metabolizing enzyme. It can be a step towards Pro production (as in Acanthamoeba castellanii [187], but it is also seen as a way to eliminate Orn in excess. Since Orn is the precursor of polyamines, involved in cell division, OAT is an actor in cell division control. This role was demonstrated in particular in Plasmodium falciparum, the agent of malaria, and probably in other Plasmodia (but not all; the OAT gene seems to be truncated in some of them) [26,188].

5.2. Fluxes in Mammals: A Subtle Compromise between Orn Degradation and Orn Synthesis The pattern in mammals is far more complex, probably because the specific pathway for Arg synthesis through N-acetylated metabolites is not functional. The same enzymes, including OAT, are thus used in both directions.

5.2.1. OAT and Orn Degradation As can be inferred from observations in other organisms, and for the various reasons given above, the main net flux (whole body) is from Orn to GSA/P5C [6,22,134,189–191]. This is also illustrated by the hyperornithinemia observed in OAT-deficient (gyrate atrophy) patients [192–194]. Conversely, OAT overexpression in transgenic mice decreases plasma and liver ornithine concentrations [195], also consistent with the ornithine catabolism direction. Some indications at the organ level of the directionality of the OAT reaction have already been given in 4.2. Briefly, in most tissues, OAT catalyzes Orn degradation. In the liver, this is a way to generate Glu for Gln synthesis and eliminate excess nitrogen, consistent with its induction by amino acids and high protein diets and its localization with Gln synthase (see 4.2). In the brain, generated Glu is a precursor for neurotransmitters. In general, in other tissues and at the whole body level, OAT is thought to remove excess Orn to avert its toxic effects. Biology 2017, 6, 18 22 of 38

5.2.2. OAT and Orn Synthesis Intestinal OAT is clearly involved in Orn synthesis to generate Arg or aliphatic polyamines. Several studies show that OAT does not work in this way in the liver, allowing the conversion of excess Orn (generated from Arg in the urea cycle) into P5C [22]. However, it is noteworthy that in the small intestine, OAT works in the reverse direction (P5C → Orn) [196], likely due to its high OAT and low PCDH content [1]. Another study supports the hypothesis that ATP and NADPH can be used to reduce Glu to glutamate-γ-semi-aldehyde, which is subsequently aminated by OAT to yield Orn in the small intestine [197,198]. The purpose of Orn synthesis by intestinal OAT differs according to the situation: to generate Arg for growth and development in early infancy, or metabolic adaptation to a fasting state (see below). Changes in tissue-specific OAT activity during growth are observed, and will be detailed below in 6.1. Briefly, in neonates, the gut generates Arg from Pro (found in maternal milk), the kidney and liver not yet being fully functional. In adults, the metabolic process is more complex: in the fasting state, or after a low protein diet/Arg-deprived diet, amino acids must be spared, and the gut produces Cit from Gln [199]. Cit is released in the portal vein and bypasses the liver, without activating the nitrogen elimination pathways (urea cycle and Gln synthesis). This is consistent with the disappearance of ASS and ASL activity in the adult gut [200]. Cit, released in the portal vein, is in turn taken up by the kidney and converted into Arg [201]. OAT plays a key role in this metabolic pathway, at the crossroads where all these sub-paths meet (Figure1), and simulation studies suggest that it may be the enzyme that controls the kinetics of the Gln to Cit conversion [202], in agreement with its up-regulation in low-protein diets. Evidence that Gln is a precursor of Arg and Cit has been reported for mice [199]. However, the fate of Orn synthesized by OAT could also be in the synthesis of polyamines, through Orn production. Experiments with 13C-labeled Gln in activated T-cells has indeed shown the appearance of labeled Orn, accounting for 30% of total Orn [114], but this pathway seems absent in resting T-cells [114]. This was also demonstrated in the proliferation of some types of tumor, by rendering the tumor independent of an external Gln supply. In both cases, OAT is a potential target for new treatments, and a patent has been registered [33]. Of note, this “reverse” pathway seems to exist only in addition to the “catabolic” one, and to date there is no example of an organism that uses OAT solely as an Orn synthesis enzyme. It therefore seems to be an evolutionary acquisition for organisms that lack a specific Arg biosynthesis pathway; it may be related to the need for discrete feeding, and so more flexibility in amino acid supply and storage, associated with pluricellular, mobile organisms like Animals.

6. OAT in Health and Disease

6.1. OAT and the Development of Mammals

6.1.1. OAT and Arg Supply in the Pre-Weaning Period As mentioned in 4.1, intestinal OAT plays a major role during development, as Arg synthesis requirements change from the fetus through weaning to adulthood. Conversion of Pro from maternal milk into Arg has been shown to be considerable in pig [203] and mouse [204] neonates, and there is evidence that human neonates also convert Pro into Arg by this pathway [205,206]. The OAT requirement seems to be all the greater as growth is fast, and newborn mice with non-functional OAT die in a few days without an Arg supply. Milk in mammals is poor in Arg, and high net Arg production by the gut is required to sustain growth [207]. Surprisingly, in contrast to what is observed in mice neonates, loss of OAT activity in human newborns (although it leads to seizures due to hyperammonemia, and slower growth) is non-lethal. This may be because of the faster relative growth rate observed in mice neonates, implying Arg requirements exceeding the availability of Arg in maternal milk [204]. The reaction can proceed in this direction because there is a large amount of GSA (in agreement with the high Pro content of maternal milk), and rapid consumption of Orn, which is converted into Biology 2017, 6, 18 23 of 38

Cit. In addition, enterocytes in newborns exhibit high ASS + ASL activities, allowing Cit conversion into Arg [208,209]. We can suggest that Orn production is high enough in neonates compared with Orn catabolism for the whole body flux of the OAT-catalyzed reaction to be reversed, with a net Orn synthesis.

6.1.2. Changes in Tissue-Specific OAT Expression

6.1.2.1. Pre-Weaning Period The pre-weaning period in rats is characterized by a high intestinal OAT activity (higher than that observed in any other rat tissue) [189]. As shown for pigs and rodents, in human neonates, OAT intestinal activity is at its maximum at birth [206], and then declines after three years to reach the adult level at five years [206]. In mice, we note an increase in liver and muscle OAT activity parallel to corticosterone levels. Thyroid hormone may also be involved, and could positively regulate OAT expression [75]. It can be postulated that during pre-weaning, a period characterized by restricted Arg supply (see above), the flux in muscle OAT reactions takes the direction of Orn synthesis in order to spare Arg [75].

6.1.2.2. Weaning Period Weaning is associated with a reduced intestinal OAT activity [204]. It is noteworthy that weaning by itself leads to a marked decrease in muscle OAT (as observed in early weaning) [75]. It can be suggested that muscle OAT activity is modulated by the transition from a milk diet (rich in lipids and relatively poor in proteins) to a diet rich in proteins [75]. Although a very high protein enrichment (up to 70 % of diet) is known to upregulate liver OAT activity, the impact of this weaning-associated dietary change on liver OAT activity is not clearly established [75,161].

6.1.2.3. Post-Weaning Period and Puberty In the post-weaning period, the onset of sex hormone secretion leads to several tissue-specific changes in OAT activity. Testosterone secretion in male mice is associated with an increase in OAT activity in the liver and to a decrease in skeletal muscle [75] and kidney [167]. The role of testosterone in the ontogenic profile in male mice is also supported in particular by the impact of orchidectomy on OAT activity in liver and muscle [75]. In rats, 17β-estradiol upregulates OAT activity in the kidney [137,164]. The impact of sex hormones on tissue-specific OAT activity could explain the marked sexual dimorphism observed. These results suggest a tissue-specific regulation of OAT activity during development. These specific features imply a fine regulation of OAT activity during development. This has been confirmed experimentally [162,189,210–213], and may occur through atRA, which increases OAT activity in Caco-2 cells through increased mRNA expression [214]. Interestingly, the presence of intestinal OAT during fetal development in pigs suggests the importance of Orn, Arg, Pro and polyamine synthesis for the development of the intestine during gestation [162]. In neonatal mice, intestinal Orn is probably not used for polyamine production [215]. Of note, several transcription factors involved in development are predicted to have binding sites in the OAT gene promoter (including PAX-4, NKX5-1, HNF1, HNF1a, En1).

6.1.3. Other Implications OAT plays an important role in Arg synthesis, but there is some evidence that OAT is involved in other aspects of development—possibly through its control of Orn availability for polyamine synthesis, such as in villous cell differentiation (see 4.7, OAT in the other tissues). During the embryo phase, a relatively high, stable level of OAT is found in the pigment epithelium of chick embryos, suggesting a significant early role of OAT in the maintenance of pigment epithelium function. Surprisingly, a decrease in OAT activity during the same period is observed in the retina Biology 2017, 6, 18 24 of 38

[AG27]. These results suggest that OAT plays a particular role in maintaining pigment epithelium and retinal function, as confirmed by the adverse impact on ocular function induced by loss of OAT activity (see 6.2). Lastly, brain regional distribution of OAT activity showed that OAT may be concentrated in glutamatergic neurons, suggesting a role of OAT for the synthesis of neurotransmitter Glu [175]. Studies in Xenopus laevis also suggest a crucial role of OAT in the control of neurogenesis during development [216].

6.2. Consequences of OAT Deficiency: Gyrate Atrophy of the Choroid and Retina (GA) OAT deficiency is the cause of a rare, serious and disabling congenital metabolic disorder characterized by gyrate atrophy (GA) of the choroid and retina [194]. Some details on causal mutations, and their consequences on the enzyme structure or activity, can be found in Section2; other mutations on non-coding regions of the gene are presented in Section3.

6.2.1. Overview As stated above, OAT deficiency in humans results in GA. To date, 65 OAT mutations have been identified according to the Human Gene Mutation Database [217]. This rare congenital metabolic disease is found worldwide, with a particularly high incidence in Finland, where its frequency is approximately 1/50,000 [218]. GA is an autosomal recessive disease clinically characterized by a childhood-onset visual disorder with myopia, followed by the development of night blindness, cataracts and progressive constriction of vision fields, leading to blindness in the fourth to fifth decade [219]. Although in the large majority of cases cognition is unimpaired, a few patients have been described with mental retardation [220]. Biologically, patients with GA display an increase in plasma Orn concentration (10–20-fold) [194], and in some cases present an overflow ornithinuria, lysinuria and cystinuria when plasma Orn exceeds 400 µmol/L [221,222]. It is noteworthy that the increase in plasma Orn occurs after the first 3 months of life [204]; the importance of this metabolic feature is underlined below.

6.2.2. Pathophysiology Although the cause of GA is well-identified, the physiopathology leading to chorioretinal degeneration is not fully understood. Current assumptions implicate the toxic effect of elevated intraocular concentrations of Orn and its metabolites in excess, such as spermine, on the retinal pigment epithelial cells [223], together with Pro deficiency in the choroid and retina [224]. The cause of mental retardation is unclear and a subject of debate. One likely hypothesis is brain creatine (Cre) deficiency secondary to Orn accumulation: high Orn concentration inhibits arginine-glycine amidinotransferase (AGAT), which results in deficient Cre synthesis [225]. It has been shown that Cre deficiency causes severe neurologic symptoms as observed in cases of guanidinoacetate methyltransferase (GAMT) deficiency. In cases of GAMT deficiency, a high-dose Cre therapy (4–8 g/day Cre monohydrate) nearly normalizes biochemical and MRS parameters, and the patients show clinical improvement [226]. However, Valayannopoulos et al. [227] have shown that mental retardation in GA seems linked to hyperornithinemia, more than to Cre deficiency itself.

6.2.3. Diagnosis In children or adults, GA diagnosis is based mainly on ocular clinical parameters. Suspicion is confirmed with increased plasma Orn levels. Firm diagnosis remains dependent on demonstrating deficient OAT activity in fibroblasts [228]. The neonatal period is a broad time window for diagnosis. Importantly, current diagnosis is usually made at the symptomatic phase, characterized by irreversible ocular damage. Diagnosis in early infancy remains unusual, and is difficult because increased plasma ammonia and urine orotic acid, found from the first few days of life, associated with reduced levels of plasma Arg and Cit can easily lead to a diagnosis of Orn carbamoyltransferase (OTC) deficiency [228]. In order to avert or Biology 2017, 6, 18 25 of 38 delay the appearance of symptoms, clinicians need an early diagnostic test (before clinical expression) to allow early management of the disease and a better prognosis. Interestingly, the 3-month window just after birth is physiologically characterized by a reversed flux in the OAT reaction (P5C → Orn) (see above for details). Thus during this period, a deficit in OAT activity leads to non-toxic subnormal plasma Orn concentrations. A neonatal diagnosis, permitting prompt treatment before irreversible damage has occurred, would offer real hope for preventing ocular and neurological impairment in GA.

6.2.4. Diagnosis in Newborns A recent study [229] highlights the usefulness (subject to confirmation in a large-scale study) of a simple diagnostic test based on the Pro/Cit ratio in plasma and a neonatal dried blood spot (NBS). The idea of using the Pro/Cit ratio in newborns resulted from a better understanding of how OAT works at this age: in early life only, there is a reversed whole body flux of the OAT reaction in the direction of Orn synthesis [204]. OAT deficiency at this age thus theoretically causes an increase in Pro and a deficiency of Orn and its products Arg and Cit (resulting in impairment of the urea cycle and attendant hyperammonemia), and so the Pro/Cit ratio increases. In practice, concentrations of plasma Orn and Arg are often subnormal in newborns with OAT deficiency: Pro is increased, while Cit is decreased [229]. This supports the use of Pro and Cit to establish a Pro/Cit ratio with the best diagnostic sensitivity (Figure 11). OAT deficiency should be added to the differential diagnosis of hyperammonemia in newborns, and the Pro/Cit ratio, which is easy to determine, could be valuable forBiology GA 2017 screening, 6, 1 and diagnosis in this neonatal period. 29 of 40

FigureFigure 11.11. Proline/citrullineProline/citrulline ratio and proline concentration in the neonatal dried blood spot ofof twotwo affectedaffected patientspatients withwith OATOAT deficiencydeficiency (case(case 11 andand casecase 2).2). TheThe solidsolid lineline indicatesindicates thethe 9999 percentilepercentile (97.6)(97.6) ofof > > 450,000 450,000 blood blood spot spot samples samples analyzed analyzed as partas part of the of Minnesotathe Minnesota NBS NBS program. program. Adapted Adapted from defrom Sain-van de Sain der-van Velden der Velden et al.[ 229et al.]. [229].

6.3. OAT Inhibition As A Possible Therapeutic Strategy 6.2.5. Therapeutic Approaches

6.3.1.Currently Potential twoUtility therapeutic of OAT Inhibitors approaches for seem the Treatment to be effective of Cancer to reduce plasma Orn concentrations below levels that cause toxic effects (400 µmol/L), and slow down the eye disease. These are an As evidenced by the live birth of OAT-null mice, OAT appears to be non-essential for cellular Arg-restricted diet [230], and vitamin B6 supplementation in cases of vitamin B6-responsive phenotype, proliferation during embryonic and fetal development [204]. However, a study conducted by Wang which concerns a minority of patients [222,231]. The link between genetic mutations and response et al. [123] has shown, at least for rapidly proliferating cancer cells, that OAT is needed to establish to treatment is still unclear. Data suggest that B6-responsiveness in human subjects depends on spindle formation; OAT was found to be over-expressed in some cancers, especially in HCC cells unidentified mechanisms other than specific OAT genotype [232]. This justifies trying vitamin B6 [234]. OAT inhibition could thus be beneficial in treating HCC or other cancers [34,235]. Currently, treatment in all GA patients and pursuing it if efficacious. one major obstacle to the development of a drug that inhibits OAT is the fear it may give rise to toxic Concerning the treatment of mental retardation, one study [233] showed the ineffectiveness of hyperornithinemia as observed in GA. However, GA is a slowly-progressing disease, and ocular Cre supplementation (1.5–2 g Cre monohydrate/day), consistent with involvement of plasma Orn lesions do not occur immediately. Hence acute treatments of cancer with OAT inhibitors should be concentration in manifesting toxic effects rather than Cre deficiency. However, the authors of this possible and safe [34].

6.3.2. Treatment of Hyperammonemia Inactivation of OAT leads to an increased endogenous level of Orn with enhancement of urea formation. This effect is similar to that observed with large doses of Arg and Orn. However, OAT inhibition seems to provide a longer-lasting protective effect against various forms of hyperammonemic states. In mice with a hereditary defect of the urea cycle, OAT inhibition leads to a decrease in ammonia concentrations in plasma and tissues and a reduced excretion of urinary orotic acid [44]. No toxic effects of Orn were found in mice. Although further studies are needed to confirm this protective effect, OAT inhibition seems of interest as an approach to treating hyperammonemia.

6.3.3. OAT in Sepsis Because of its place at the crossroads of Arg and Gln synthesis, it has been suggested that OAT could play a role in the adaptive response in early endotoxemia. Ventura et al [156] showed that the adaptive response of nitrogen metabolism in early endotoxemia is associated with a physiological inhibition of liver OAT, the latter being responsible for increased Orn in the liver. This OAT inhibition could be considered as a protective mechanism, sparing Orn for other metabolic pathways (Pro and polyamine synthesis). However, in this study, OAT seems to be non-limiting for Gln synthesis under physiological and endotoxemic conditions.

7. Conclusions The importance of OAT is abundantly illustrated by the consequences of its mutation, which leads either to unviable animal newborns (unless they receive a special diet) or to gyrate atrophy, in humans. OAT is a highly conserved enzyme among eukaryotes, and is involved in amino acid

Biology 2017, 6, 18 26 of 38 study suggest testing larger doses of Cre (as for GAMT deficiency) or initiating the treatment very early [227]. Moreover, the reduction in plasma Orn levels achieved by an Arg-restricted diet do not lead to an increase in plasma Cre levels, probably because Arg is the precursor of Cre [227]. Specifically, in children younger than 3 months of age, Arg intake should not be restricted until plasma Orn begins to increase. It is noteworthy that management of an Arg-restricted diet requires monitoring of growth, nutritional status and plasma amino acid levels.

6.3. OAT Inhibition As A Possible Therapeutic Strategy

6.3.1. Potential Utility of OAT Inhibitors for the Treatment of Cancer As evidenced by the live birth of OAT-null mice, OAT appears to be non-essential for cellular proliferation during embryonic and fetal development [204]. However, a study conducted by Wang et al. [123] has shown, at least for rapidly proliferating cancer cells, that OAT is needed to establish spindle formation; OAT was found to be over-expressed in some cancers, especially in HCC cells [234]. OAT inhibition could thus be beneficial in treating HCC or other cancers [34,235]. Currently, one major obstacle to the development of a drug that inhibits OAT is the fear it may give rise to toxic hyperornithinemia as observed in GA. However, GA is a slowly-progressing disease, and ocular lesions do not occur immediately. Hence acute treatments of cancer with OAT inhibitors should be possible and safe [34].

6.3.2. Treatment of Hyperammonemia Inactivation of OAT leads to an increased endogenous level of Orn with enhancement of urea formation. This effect is similar to that observed with large doses of Arg and Orn. However, OAT inhibition seems to provide a longer-lasting protective effect against various forms of hyperammonemic states. In mice with a hereditary defect of the urea cycle, OAT inhibition leads to a decrease in ammonia concentrations in plasma and tissues and a reduced excretion of urinary orotic acid [44]. No toxic effects of Orn were found in mice. Although further studies are needed to confirm this protective effect, OAT inhibition seems of interest as an approach to treating hyperammonemia.

6.3.3. OAT in Sepsis Because of its place at the crossroads of Arg and Gln synthesis, it has been suggested that OAT could play a role in the adaptive response in early endotoxemia. Ventura et al [156] showed that the adaptive response of nitrogen metabolism in early endotoxemia is associated with a physiological inhibition of liver OAT, the latter being responsible for increased Orn in the liver. This OAT inhibition could be considered as a protective mechanism, sparing Orn for other metabolic pathways (Pro and polyamine synthesis). However, in this study, OAT seems to be non-limiting for Gln synthesis under physiological and endotoxemic conditions.

7. Conclusions The importance of OAT is abundantly illustrated by the consequences of its mutation, which leads either to unviable animal newborns (unless they receive a special diet) or to gyrate atrophy, in humans. OAT is a highly conserved enzyme among eukaryotes, and is involved in amino acid metabolism. In most organisms, it is involved in the conversion of ornithine into glutamate or proline. However, in mammals, it is also involved in ornithine synthesis, depending on the organ and the development stage. This ability to switch from net ornithine degradation to net ornithine synthesis is associated with a fine regulation of its kinetic properties, and of its expression. As described above, this regulation results from the combination of several factors, ranging from the concentration of the OAT and its pH environment, to its transcription level, with a subtle balance between a background, non-specific expression, and a tissue-specific regulation by targeted transcription factors or miRNA. The huge amounts of available bioinformatics and “omics” data suggest that OAT is a player in a complex network of interacting proteins, either at the protein level or at the DNA expression level. Biology 2017, 6, 18 27 of 38

Although the main metabolic roles of OAT are now quite well understood, they do not explain all the reported observations—for instance, the relationship between OAT mutation and the pathophysiology of gyrate atrophy remains poorly understood. Similarly, OAT involvement in pathologies such as cancer, and its detection in the cell nucleus suggest that it could be involved in the regulation of several cell processes, probably by allowing a local control of ornithine, glutamate and related small molecules (such as polyamines, neutrotransmitters) involved in controlling the balance between the main cell function pathways. Research is still needed to gain a better understanding of these aspects, and of how apparently unrelated observations concerning OAT can be linked.

Acknowledgments: This paper is dedicated to the memory of Prof Simone Bénazeth (1944–2014). We thank Isabelle Ladriere for expert secretarial assistance, and Prof Stephen Bearne (Dalhousie University, Halifax, NS, Canada) for clarifying the hydrate/aldehyde GSA equilibrium. Conflicts of Interest: The authors declare no conflict of interest.

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