Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bond

Abstract

Cystathionine β-synthase (CBS) is the central enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of the three major enzymes involved in the biogenesis of H 2 S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a C 272 XXC 275 motif whose function has yet to be determined. In the present study, we demonstrated that the CXXC motif exists in oxidized and reduced states in the recombinant enzyme by mass spectroscopic analysis and a thiol labeling assay. The activity of reduced CBS is ~2-to 3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys 272 -Cys 275 disulfide bond in CBS has a midpoint potential of -314 mV at pH 7.4. Additionally, the CXXC motif also exists in oxidized and reduced states in human embryonic kidney 293 (HEK293) cells under oxidative and reductive conditions, and stressing these cells with dithiothreitol (DTT) results in more reduced enzyme and a concomitant increase in H 2 S production in live HEK293 cells as determined using a H 2 S fluorescent probe. By contrast, incubation of cells with aminooxyacetic acid (AOAA), an inhibitor of CBS and cystathionine γ-lyase (CSE), eliminates the increase of H 2 S production after the cells were exposed to DTT. These findings indicate that CBS is post-translationally regulated by a redox-active disulfide bond in the CXXC motif. The results als o demonstrate that CBS-derived H 2 S production is increased in cells under reductive stress conditions.

Maternal metabolic profile predicts high or low risk of an autism pregnancy outcome

“…The cystine/cysteine redox couple is considered to be a better indication of plasma oxidative stress than the GSH/GSSG ratio which is the dominant intracellular redox couple (Jones, 2006b). Cystathionine beta synthase (CBS), the enzyme that metabolizes homocysteine to initiate transsulfuration, is regulated by a redox sensitive cysteine that reduces CBS activity when oxidized (Niu et al, 2018). The increase in oxidized cystine/cysteine in High Risk mothers is consistent with a redox responsive decrease in CBS activity that could contribute to the increase in homocysteine (Figure 1).…”

Assessing computational predictions of the phenotypic effect of cystathionine‐beta‐synthase variants

“…The p.A361T mutant could therefore potentially interfere with a functionally relevant modification (e.g., S‐nitrosylation) of p.C370. Similarly, modification of p.A288 could disrupt the pairing or orientation between beta5 and beta6, thereby potentially impacting the 272‐CxxC‐275 oxygen sensing motif of CBS, a redox active disulfide bond that allosterically controls CBS activity (Niu et al, ).…”

Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine

“…Interestingly, when embryos were treated with a combination of lanthionine and GSH (at low GSH dosage; up to 50 µM), we could observe a typical additive effect, while, as GSH concentrations increased in the medium, the effect could rather be consistent with a synergistic model of action. This may be explained by the fact that transsulfuration enzymes, particularly CBS, are influenced by the oxidative microenvironment in many ways, also including a variation in the concentration of GSH [ 36 , 37 ].…”

“…However, at molecular level, the effects of lanthionine were remarkably evident even in the presence of GSH and appeared to be influenced by GSH in a more complex way. A further additional allosteric regulatory mechanism may depend on conformational changes induced by formation of a redox active disulfide bond in human CBS, and reversal by DTT, which has been recently shown to occur [ 37 ].…”