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Purification and properties of cystathionine beta-lyase from Arabidopsis thaliana overexpressed in Escherichia coli.

ABSTRACT: Cystathionine beta-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5'-phosphate-dependent beta-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine beta-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine beta-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5'-phosphate. Interaction between the apoenzyme and pyridoxal 5'-phosphate is extremely tight, being characterized by a Kd value of 0.5 microM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine beta-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine beta-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5'-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.

SUBMITTER: Ravanel S

PROVIDER: S-EPMC1217943 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Project description:Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H2S) and glutathione (GSH) may play an important role in ameliorating disease progression. H2S production is driven by cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H2S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primate (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and Mϋller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser capture microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H2S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.

Project description:Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)‑induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild‑type (WT) mice and in mice with reduced expression of one of the three H2S‑producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S‑producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue‑dependent upregulation of some of the H2S‑producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE‑/‑ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/‑ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/‑, CSE‑/‑ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/‑ and CSE‑/‑ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)‑1β, IL‑6, IL‑10, IL‑12 and interferon (IFN)γ] in plasma; TNFα, IL‑10 and IL‑12 levels tended to be lower in all three groups of animals expressing lower levels of H2S‑producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

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Purification and properties of cystathionine beta-lyase from Arabidopsis thaliana overexpressed in Escherichia coli.

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