Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role.

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.     

35S]Lanthionine ketimine binding to bovine brain membranes

2H-1,4-Thiazine-5,6-dihydro-3,5-dicarboxylic acid (trivial name: lanthionine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts. This compound has been synthesized in a heavily labeled form starting from L-[35S]cysteine and purified by high performance liquid chromatography. We demonstrate the existence of a saturable and reversible binding of [35S]lanthionine ketimine to bovine brain membranes. A single population of binding sites with a concentration of 260 +/- 12 fmol/mg protein and a dissociation constant of 58 +/- 14 nM is present. Specific binding is competitively inhibited by other structurally similar imino acids, namely S-aminoethyl-L-cysteine ketimine and cystathionine ketimine. These results suggest a possible functional role for these ketimines in nervous system.     

Imine Reductases: A Comparison of Glutamate Dehydrogenase to Ketimine Reductases in the Brain

A key intermediate in the glutamate dehydrogenase (GDH)-catalyzed reaction is an imine. Mechanistically, therefore, GDH exhibits similarities to the ketimine reductases. In the current review, we briefly discuss (a) the metabolic importance of the GDH reaction in liver and brain, (b) the mechanistic similarities between GDH and the ketimine reductases, (c) the metabolic importance of the brain ketimine reductases, and (d) the neurochemical consequences of defective ketimine reductases. Our review contains many historical references to the early work on amino acid metabolism. This work tends to be overlooked nowadays, but is crucial for a contemporary understanding of the central importance of ketimines in nitrogen and intermediary metabolism. The ketimine reductases are important enzymes linking nitrogen flow among several key amino acids, yet have been little studied. The cerebral importance of the ketimine reductases is an area of biomedical research that deserves far more attention.     

Detection of cystathionine and lanthionine ketimines in human urine

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.     

Detection of 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine) in the bovine brain by a fluorometric assay

A new sulfur imino acid, 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine), has been detected in the bovine brain by means of fluorometric and HPLC procedures. The fluorometric assay is based on the fluorescent property of the copper-ketimine interaction product at pH 11.5. Other ketimines do not fluoresce in these conditions. The fluorophore exhibits an excitation maximum at 353 nm and an emission at 462 nm and is stable for at least 24 h. In the test conditions the fluorescence is proportional to the ketimine concentration from 1 to 200 microM. Detection of endogenous lanthionine ketimine has been performed after a simple enrichment procedure (brain deproteinization and extraction with diethyl ether) which minimizes degradative by-reactions of the unstable ketimine. The concentration of this new sulfur imino acid in the brain ranges from 0.5 to 1 nmol/g in three different samples. Identification and quantitations were confirmed by an HPLC procedure which takes advantage of the selective absorption at 380 nm of the phenylisothiocyanate-ketimine adduct. The identification of lanthionine ketimine in nervous tissues may have important metabolic and physiological implications.     

Bovine brain ketimine reductase

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.     

Oxime-metabolizing activity of liver aldehyde oxidase

Liver aldehyde oxidase in the presence of its electron donor exhibited a significant oxime-metabolizing activity toward some different types of oximes under anaerobic conditions. Acetophenone oxime and salicylaldoxime were exclusively converted to the corresponding oxo compounds, whereas benzamidoxime was converted to the corresponding ketimine. With d-camphor oxime, the formation of both the corresponding oxo compound and ketimine was observed. Stoichiometric studies showed that the formation of oxo compounds is accompanied by nearly equimolar ammonia. We propose a mechanism of oxime biotransformation that liver aldehyde oxidase catalyzes the reduction of oximes to the corresponding ketimines which in turn undergo, depending on their chemical stability, nonenzymatic hydrolysis to the corresponding oxo compounds and ammonia.     

Influence of diet on cystathionine ketimine and lanthionine ketimine content in human urine

A simple HPLC procedure for the routine analyses of Cystathionine ketimine (CK) and Lanthionine ketimine (LK) content in human urine has been developed. The values obtained in morning urine of fifteen healthy subjects (both sexes, 25-45 years old) on a common mixed diet are 330-2480 micrograms/g creatinine (mean 1110) of CK and 100-420 micrograms/g creatinine (mean 200) of LK. Quantitation of the two ketimines in urine of subjects on strictly vegetarian diet indicate that while the excretion of LK is independent of the diet, the excretion of CK is significantly decreased in conditions of vegetarian diet.     

Comparative mechanisms of vitamin B6-catalyzed beta-decarboxylation and beta-dephosphonylation in model systems

Reaction pathways and mechanisms of vitamin B6-catalyzed beta-decarboxylation and beta-dephosphonylation of aminocarboxylic and aminophosphonic acids in model systems are compared. It was found that both reactions require prior transamination of an aldimine intermediate to a ketimine. For ketimines having carboxylate or phosphonate groups substituted on the beta-carbon atoms of the keto acid residue, there is a hydrogen ion or metal ion-activated covalent bond pathway which involves a shift of electron pairs toward the coordinated ketimine nitrogen, leading to beta-gamma, C-C or C-P bond fission and release of carbon dioxide or metaphosphate, respectively. Comparison of these reactions indicates that beta-decarboxylation is 10(6) faster than the corresponding dephosphonylation reaction. Since only a few studies of vitamin B6-catalyzed dephosphonylation have been carried out, suggestions are made for further studies with substrates designed to elucidate the reaction mechanisms involved.     

Alternative functions of the brain transsulfuration pathway represent an underappreciated aspect of brain redox biochemistry with significant potential for therapeutic engagement

Scientific appreciation for the subtlety of brain sulfur chemistry has lagged, despite understanding that the brain must maintain high glutathione (GSH) to protect against oxidative stress in tissue that has both a high rate of oxidative respiration and a high content of oxidation-prone polyunsaturated fatty acids. In fact, the brain was long thought to lack a complete transsulfuration pathway (TSP) for cysteine synthesis. It is now clear that not only does the brain possess a functional TSP, but brain TSP enzymes catalyze a rich array of alternative reactions that generate novel species including the gasotransmitter hydrogen sulfide (H₂S) and the atypical amino acid lanthionine (Lan). Moreover, TSP intermediates can be converted to unusual cyclic ketimines via transamination. Cell-penetrating derivatives of one such compound, lanthionine ketimine (LK), have potent antioxidant, neuroprotective, neurotrophic, and antineuroinflammatory actions and mitigate diverse neurodegenerative conditions in preclinical rodent models. This review will explore the source and function of alternative TSP products, and lanthionine-derived metabolites in particular. The known biological origins of lanthionine and its ketimine metabolite will be described in detail and placed in context with recent discoveries of a GSH- and LK-binding brain protein called LanCL1 that is proving essential for neuronal antioxidant defense; and a related LanCL2 homolog now implicated in immune sensing and cell fate determinations. The review will explore possible endogenous functions of lanthionine metabolites and will discuss the therapeutic potential of lanthionine ketimine derivatives for mitigating diverse neurological conditions including Alzheimer׳s disease, stroke, motor neuron disease, and glioma.     

The conformation of catalytically functional Schiff's bases: the pyruvate--lysine ketimine of 2-keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida

The reduction stereochemistry of the Schiff's base formed between pyruvate and the ?-amino of the catalytic lysine of 2-keto-3-deoxygluconate-6-P-phosphate aldolase of Pseudomonas putida was investigated. Reduction was stereoselective yielding 55.73% N⁶-[(1R)-and 44.27% N⁶-[(1S)-1-carboxyethyl]-S-lysine. Thus the reducing agent predominated slightly at the si face of the ketimine carbon. For comparison, the reduction stereochemistry of the pyruvate-lysine ketimine formed on d-amino acid oxidase during d-alanine turnover at pH 8.5 was also investigated. In this case reduction was random, consistent with nonactive site participation in that transimination reaction generating the ketimine, as postulated by other investigators of this enzyme.     

A novel enantioselective synthesis of (S)-(-)- and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template

An asymmetric synthesis of the optically pure isomers of the minor tobacco alkaloid and CNS nicotine metabolite, nornicotine, has been achieved with moderately high optical purity. The synthetic pathway involves alkylation of a chiral ketimine, prepared from either 1R,2R,5R-(+)- or 1S,2S,5S-(-)-2-hydroxy-3-pinanone and 3-(aminomethyl)pyridine with 3-bromopropan-1-ol. After cleavage of the respective C-alkylated ketimines with NH2OH.HCl, and treatment of the resulting amino alcohols with HBr, followed by base-catalyzed intramolecular ring closure, (S)-(-)-nornicotine and (R)-(+)-nornicotine were obtained with ee values of 91% and 81%, respectively.     

Reciprocal Control of Thyroid Binding and the Pipecolate Pathway in the Brain

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of –C=N– (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ?¹-piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main l-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in l-ornithine/l-glutamate/l-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.     

The role of glutamine transaminase K (GTK) in sulfur and alpha-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants

Glutamine transaminase K (GTK), which is a freely reversible glutamine (methionine) aromatic amino acid aminotransferase, is present in most mammalian tissues, including brain. Quantitatively, the most important amine donor in vivo is glutamine. The product of glutamine transamination (i.e., alpha-ketoglutaramate; alphaKGM) is rapidly removed by cyclization and/or conversion to alpha-ketoglutarate. Transamination is therefore "pulled" in the direction of glutamine utilization. Major biological roles of GTK are to maintain low levels of phenylpyruvate and to close the methionine salvage pathway. GTK also catalyzes the transamination of cystathionine, lanthionine, and thialysine to the corresponding alpha-keto acids, which cyclize to ketimines. The cyclic ketimines and several metabolites derived therefrom are found in brain. It is not clear whether these compounds have a biological function or are metabolic dead-ends. However, high-affinity binding of lanthionine ketimine (LK) to brain membranes has been reported. Mammalian tissues possess several enzymes capable of catalyzing transamination of kynurenine in vitro. Two of these kynurenine aminotransferases (KATs), namely KAT I and KAT II, are present in brain and have been extensively studied. KAT I and KAT II are identical to GTK and alpha-aminoadipate aminotransferase, respectively. GTK/KAT I is largely cytosolic in kidney, but mostly mitochondrial in brain. The same gene codes for both forms, but alternative splicing dictates whether a 32-amino acid mitochondrial-targeting sequence is present in the expressed protein. The activity of KAT I is altered by a missense mutation (E61G) in the spontaneously hypertensive rat. The symptoms may be due in part to alteration of kynurenine transamination. However, owing to strong competition from other amino acid substrates, the turnover of kynurenine to kynurenate by GTK/KAT I in nervous tissue must be slow unless kynurenine and GTK are sequestered in a compartment distinct from the major amino acid pools. The possibility is discussed that the spontaneous hypertension in rats carrying the GTK/KAT I mutation may be due in part to disruption of glutamine transamination. GTK is one of several pyridoxal 5'-phosphate (PLP)-containing enzymes that can catalyze non-physiological beta-elimination reactions with cysteine S-conjugates containing a good leaving group attached at the sulfur. These elimination reactions may contribute to the bioactivation of certain electrophiles, resulting in toxicity to kidney, liver, brain, and possibly other organs. On the other hand, the beta-lyase reaction catalyzed by GTK may be useful in the conversion of some cysteine S-conjugate prodrugs to active components in vivo. The roles of GTK in (a) brain nitrogen, sulfur, and aromatic amino acid/kynurenine metabolism, (b) brain alpha-keto acid metabolism, (c) bioactivation of certain electrophiles in brain, (d) prodrug targeting, and (e) maintenance of normal blood pressure deserve further study.     

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.     

New enzymatic changes of L-cystathionine catalyzed by bovine tissue extracts

L-Cystathionine was used as substrate for enzyme systems prepared by heating bovine tissue extracts in the presence of pyruvate at 60 degrees C for 10 min. Analysis of the products indicated that the systems converted L-cystathionine into the cyclic ketimine form which was detected by its spectral properties and by chromatography on the amino acid analyzer. Alanine, alpha-aminobutyrate and cystine were also produced. Pyruvate and alpha-ketobutyrate enhance the production of the ketimine by liver, kidney and heart extracts, and are necessary for the brain extracts: alpha-Ketoglutarate is much less effective and its presence favors the production of homocystine by all the extracts. Homocystine was found in the brain incubates when any of the ketoacids assayed were added. The overall reaction is explained by the action of heat stable cystathionine gamma-lyase and beta-synthase which produce alpha-ketobutyrate and pyruvate used for the transamination of the remaining cystathionine to the monoketoacid. This last compound cyclizes spontaneously into the ketimine form thus avoiding the removal of the second amino group. This represents a new nontransulfurative path leading to the production of a seven membered etherocyclic product whose biochemical implications are yet unexplored.     

Lysine metabolism in mammalian brain: an update on the importance of recent discoveries

The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ?¹-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ?¹-piperideine-2-carboxylate (P2C) and its reduced metabolite l-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to l-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [μ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3′-triiodothyronine (T₃) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.     

Detection of Cystathionine Ketimine and Lanthionine Ketimine in Human Brain

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.     

Aspartate aminotransferase catalyzed oxygen exchange with solvent from oxygen-18-enriched alpha-ketoglutarate: evidence for slow exchange of enzyme-bound water

Partitioning of the ketimine (or ketimine + quinonoid) intermediate(s) in the mitochondrial aspartate aminotransferase reactions was investigated by following the rates of loss of 18O from carbonyl-18O-enriched alpha-ketoglutarate together with the rate of L-glutamate formation. The ratio of these rate constants was found to equal 1 at 10 degrees C, implying that the above intermediate(s) face(s) equal barriers with respect to the forward and reverse reactions. This partition ratio of 1 together with that measured from the alpha-amino acid side of the reaction [Julin, D.A., Wiesinger, H., Toney, M. D., & Kirsch, J.F. (1989) Biochemistry (preceding paper in this issue)] suggests that the rate constant for exchange of alpha-ketoglutarate-derived H2(18)O from the ketimine (or ketimine + quinonoid) form(s) of the enzyme with solvent is comparable with that for kcat.     

The oxidation of sulfur containing cyclic ketimines The sulfoxide is the main product of S-aminoethyl-cysteine ketimine autoxidation

Summary The products of autoxidation of S-aminoethyl-L-cysteine ketimine (AECK) have been analysed with the amino acid analyzer, with thin layer chromatography and with high performance liquid chromatography. Under the conditions of the assay (pH 8.5, 38°C, O₂ bubbling) AECK is almost totally oxidized in 1.5 hours. Among the final products a component running fast in HPLC, named Cx1, has been isolated, reduced with NaBH₄ and analysed. Reduced Cx1 resulted to show the same properties of synthetic thiomorpholine-3-carboxylic acid-S-oxide, known in the past literature with the name of chondrine. On the basis of these results and by specific chromatographic tests, Cx1 has been identified as the sulfoxide of AECK. Among the other autoxidation products, thiomorpholine-3-one has been identified. The detection, after HCl hydrolysis, of glyoxylic acid and mesoxalic semialdehyde together with cysteamine indicates that compounds provided with easily cleavable S-C bonds, possibly thiohemiacetals or (and) thioesters, are the likely intermediates for other products. AECK sulfoxide and thiomorpholine-3-one are relatively stable and cannot be taken as the main intermediates for the remaining oxidation products.Abbreviations AAA amino acid analyzer - TLC thin layer chromatography - HPLC high performance liquid chromatography - AECK S-aminoethyl-L-cysteine ketimine - AECK-SO aminoethylcysteine ketimine sulfoxide - TMA thiomorpholine-3-carboxylic acid - TMA-SO thiomorpholine-3-carboxylic acid-S-oxide - CMCA S-carboxymethylcysteamine - DNPH 2,4-dinitrophenylhydrazine