Cysteine Sulphinic Acid Decarboxylase Research Articles

Hepatic cysteine sulphinic acid decarboxylase depletion and defective taurine metabolism in a rat partial nephrectomy model of chronic kidney disease

BackgroundTaurine depletion occurs in patients with end-stage chronic kidney disease (CKD). In contrast, in the absence of CKD, plasma taurine is reported to increase following dietary L-glutamine supplementation. This study tested the hypothesis that taurine biosynthesis decreases in a rat CKD model, but is rectified by L-glutamine supplementation.MethodsCKD was induced by partial nephrectomy in male Sprague-Dawley rats, followed 2 weeks later by 2 weeks of 12% w/w L-glutamine supplemented diet (designated NxT) or control diet (NxC). Sham-operated control rats (S) received control diet.ResultsTaurine concentration in plasma, liver and skeletal muscle was not depleted, but steady-state urinary taurine excretion (a measure of whole-body taurine biosynthesis) was strongly suppressed (28.3 ± 8.7 in NxC rats versus 78.5 ± 7.6 μmol/24 h in S, P < 0.05), accompanied by reduced taurine clearance (NxC 0.14 ± 0.05 versus 0.70 ± 0.11 ml/min/Kg body weight in S, P < 0.05). Hepatic expression of mRNAs encoding key enzymes of taurine biosynthesis (cysteine sulphinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO)) showed no statistically significant response to CKD (mean relative expression of CSAD and CDO in NxC versus S was 0.91 ± 0.18 and 0.87 ± 0.14 respectively). Expression of CDO protein was also unaffected. However, CSAD protein decreased strongly in NxC livers (45.0 ± 16.8% of that in S livers, P < 0.005). L-glutamine supplementation failed to rectify taurine biosynthesis or CSAD protein expression, but worsened CKD (proteinuria in NxT 12.5 ± 1.2 versus 6.7 ± 1.5 mg/24 h in NxC, P < 0.05).ConclusionIn CKD, hepatic CSAD is depleted and taurine biosynthesis impaired. This is important in view of taurine’s reported protective effect against cardio-vascular disease - the leading cause of death in human CKD.

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

Taurine

Taurine, a sulfur-containing amino acid (Figure), has been termed a functional nutrient that could be used to protect against, among others, diabetes mellitus and atherosclerosis.1 Indeed, an increasing body of literature supports the use of taurine supplements. Because taurine has very diverse functions, notably, intracellular osmoregulation and bile acid formation, and is abundantly present in several organs, multiple pathways could be involved. Some of these are discussed in this editorial. Figure. A simplified biosynthesis pathway from methionine to taurine. Methionine is converted to homocysteine in 3 steps. Homocysteine is used as a substrate to form cysteine in 2 steps but can also be converted back to methionine by 2 different mechanisms. Cysteine, precursor for the important antioxidant glutathione and the gaseous transmitter/signaling molecule H2S, can be further converted to cysteine sulfinate. Cysteine sulfinate is converted to taurine in 2 steps via 2 different pathways. Taurine can decrease methionine by decreasing uptake. Whether taurine decreases homocysteine directly or via a reduction of methionine is unclear. Of the 20 canonical amino acids, 18 are composed of carbon, hydrogen, nitrogen, and oxygen only. The remaining 2, methionine and cysteine, also contain 1 atom of sulfur. Because sulfur is not as electronegative as oxygen, the sulfur-containing amino acids play a key role in protein structure and synthesis.2 Methionine and cysteine also play important roles in cell metabolism. For instance, methionine serves as a substrate for S-adenosylmethionine, which is vital for methylation of nucleic acids, proteins, lipids, etc. In proteins, cysteine easily forms double bonds with other cysteine residues, thus determining tertiary structure and binding sites. Moreover, cysteine is substrate for glutathione, an important intracellular antioxidant, and H2S, a gas that can induce endothelial-dependent relaxation.3 Cysteine is considered a nonessential amino acid because it is synthesized …

Stereochemistry of cysteinesulphinic acid decarboxylase

An h.p.i.c.-based assay for cysteinesulphinic acid decarboxylase has been developed. The assay simultaneously measures glutamate decarboxylation and/or taurine formation. Using the cysteinesulphinic acid decarboxylase in rat liver homogenate, L-[2-2H1]cysteinesulphinic acid was converted into [2-2H1]hypotaurine. Similarly, unlabelled L-cysteinesulphinic acid was transformed by rat liver homogenate in a D2O-containing medium into the enantiomeric [2-2H1] hypotaurine. The products were desulphurized by treatment with Raney nickel, and the resulting enantiomeric [1-2H1]ethylamines converted into the corresponding (–)-camphanamides. Deuterium n.m.r. spectra of the latter revealed that the decarboxylations proceeded stereospecifically with retention of configuration.

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes.

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.

Developmental pattern of cysteine sulphinic acid transaminase activity in some areas of mice nervous system.

Abstract Cysteine sulphinic acid (CSA), a key intermediate in the major pathway of taurine metabolism, is catabolised by cysteine sulphinic acid decarboxylase (CSA-D) to hypotaurine and by the cysteine sulphinic acid transaminase (CSA-T) to glutamate. To continue earlier studies about the latter enzyme, the CSA-T activity was measured in the developing olfactory bulb, cerebellum, whole brain and retina of C57/B16 and C3H/He inbred mice. Differences in the developmental pattern between these two strains of mice in the retinae and in the olfactory bulb were observed. The significance of the differences is discussed.

Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain.

Abstract—Certain of the sulphur containing amino acids have been associated with synaptic transmission in the central nervous system. The enzymes involved in the synthesis of these putative neurotransmitter or modulator compounds have a different subcellular distribution in rat brain from those enzymes that catalyse the synthesis of other compounds in this pathway. Methionine adenosyltransferase and 5‐methyltetrahydrofolate‐homocysteine methyltransferase catalyse reactions that maintain the methylation functions of the pathway and are found in soluble fractions. Cystathionine β‐synthase, cystathionase, cysteine dioxygenase and cysteine sulphinic acid decarboxylase catalyse the synthesis of those sulphur‐containing amino acids implicated in neurotransmitter functions and these enzymes have both paniculate and soluble components. Serine hydroxymethyltransferase, which also has a particulate fraction in brain, is responsible for the synthesis of the neurotransmitter glycine, in addition to its role in the methionine‐related metabolism of folate.

Formation and excretion of taurine in x-irradiated rats.

SummaryUrinary excretion profiles of taurine have been estimated in male Wistar rats exposed to doses of x-rays ranging from 100–1000 R. Maximum levels were observed in urine collected 24 hours after irradiation. Urinary taurine returned to normal values within 48 hours only in rats which had received sublethal doses (100–400 R). The high taurine levels persisted even after 72 hours in rats receiving more than LD50, i.e. 600–1000 R. The dose–response relationship obtained indicates a sort of a threshold dose beyond which the recovery mechanisms apparently do not operate.Urinary excess of taurine could be ascribed to enhanced liver cysteic acid decarboxylase activity. The increase in enzyme activity corresponds to the maximum appearance of taurine in the urine. Cysteine sulphinic acid decarboxylase did not show any variation.Formation of taurine in vivo was also followed up after intraperitoneal injection of cysteine-3-14C. There was enhanced recovery of radioactivity of the injected amino acid in blood and urine of x-irradiated animals, indicating increased synthesis of taurine. Hypertaurinuria could be prevented by prior treatment with desoxypyridoxine.

Activities of cystathionase, cysteine sulphinic acid decarboxylase and serine dehydrase in the liver of tumour-bearing rats.

Dans le foie de rats porteurs de tumeurs (epitheliome de Guerin et sarcome de l'estomac), les activites de la cystathionase, de la decarboxylase de l'acide cysteine sulfinique et de la serine dehydrase sont considerablement diminuees: cette diminution est de l'ordre de 50%, 15 jours apres implantation de ces tumeurs. Si a ce moment le sarcome (qui ne provoque pas de metastases) est chirurgicalement extirpe, on observe une restauration des activites enzymatiques qui, apres 15 jours, est totale pour la cystathionase et tres significative pour la decarboxylase.

Effects of puromycin and actinomycin D on the increase of cystathionase and cysteine sulphinic acid decarboxylase activities in the liver of thyroidectomized rat.

PREVIOUS investigations carried out in this laboratory have shown that the concentration of two cytoplasmic rat liver enzymes dependent on pyridoxal phosphate, namely, cysteine sulphinic acid (CSA) decarboxylase (L. cysteine sulphinate carboxylase E.C. 4.1.1.29) and cystathionase (L. homoserine hydrolyase E.C. 4.2.1.15), are adaptively increased in thyroidectomized animals1,2. The events underlying these changes in response to thyroidectomy are not known; however, we have suggested that the increase of activities may be attributable to an increased biosynthesis of the two apoenzymes3, and recently we have obtained results which agree with such a hypothesis4. The experiments reported here were undertaken in order to determine whether these increases are prevented by injections of inhibitors of protein biosynthesis—puromycin5—and of the DNA-directed RNA synthesis—actinomycin D (ref. 6). Both experiments were carried out in similar conditions except that in the first rats were given intraperitoneal injections of puromycin daily for 4 days at a dose each of 5 mg/100 g of body weight, whereas in the second actinomycin D was injected by the same route and for the same time at a dose of 3 µg/100 g of body weight. The animals were killed 4 h after the last injection. The amounts of inhibitor injected were chosen taking into consideration the report of Weber et al.7 and the results of preliminary assays.

Influence d'injections de thyroxine sur la décarboxylation de l'acide l-glutamique par le cerveau du rat et sur celle de l'acide l-cystéinesulfinique par le foie du rat

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

Amino acid decarboxylases in rat brain and liver

1. 1. Liver cysteine sulphinic acid decarboxylase is found in the clear supernatant of a liver suspension after fractional centrifugation, while brain cysteine sulphinic acid decarboxylase is more or less associated with particulate matter. 2. 2. Thiol groups and pyridoxal phosphate are shown to be necessary for the activity of liver and brain decarboxylases. 3. 3. Further evidence is presented to suggest that cysteine sulphinic and cysteic acids are decarboxylated by the same enzyme in the liver. 4. 4. Evidence is also given that glutamic, cysteine sulphinic and cysteic acids are decarboxylated by the same enzyme in the brain.

The mechanism of the inhibition of decarboxylases by ISOnicotinyl hydrazide

1. 1. The kinetics of inhibition of liver cysteine sulphinic acid decarboxylase by isonicotinyl hydrazide and hydroxylamine have been compared. 2. 2. Evidence is presented to suggest that decarboxylase inhibition is the result of interaction between the formyl group of pyridoxal phosphate and the inhibitor. 3. 3. It is suggested that the rate of inhibition of enzymes by isonicotinyl hydrazide may be used to demonstrate the presence of pyridoxal phosphate as a prosthetic group.