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 H₂S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a ²⁷²CXXC²⁷⁵ 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 ∼2–3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys²⁷²–Cys²⁷⁵ 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 HEK293 cells under oxidative and reductive conditions, and stressing these cells with DTT results in more reduced enzyme and a concomitant increase in H₂S production in live HEK293 cells as determined using a H₂S fluorescent probe. By contrast, incubation of cells with aminooxyacetic acid, an inhibitor of CBS and cystathionine γ-lyase, eliminated the increase of H₂S 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 also demonstrate that CBS-derived H₂S production is increased in cells under reductive stress conditions.

Endogenous hydrogen sulfide (H₂S), which is primarily generated by 3-mercaptopyruvate sulfurtransferase (3-MST) in Escherichia coli (E. coli) under aerobic conditions, renders bacteria highly resistant to oxidative stress. However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H₂S in E. coli under anaerobic conditions. A significant decrease in H₂S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (ΔmstA). Furthermore, the H₂S-producing activity of recombinant IscS using L-cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H₂S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be −329 ± 1.6 mV. Moreover, in E. coli cells H₂S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (ΔiscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 μM Na₂S (an H₂S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 μM Na₂S to culture medium stimulates ATP synthesis in the mutant E. coli (ΔiscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H₂S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions.

A high-throughput assay was developed to identify inhibitors of cystathionine β-synthase (CBS), which is one of three key enzymes involved in H₂S biosynthesis. Screening of 6491 natural compounds identified several selective CBS inhibitors, which suppressed the proliferation of HT29 cancer cells, with IC₅₀ values of less than 10 μM.

Hydrogen sulfide (H₂S), which is generated mainly by cystathionine γ-lyase (CSE) in the cardiovascular system, plays a pivotal role in a wide range of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is poorly understood. Herein, we show that oxidation induces the disulfide bond formation between Cys252 and Cys255 in the CXXC motif, thus stimulating the H₂S-producing activity of CSE. The activity of oxidized CSE is approximately 2.5 fold greater than that of the reduced enzyme. Molecular dynamics and molecular docking suggest that the disulfide bond formation induces the conformational change in the active site of CSE and consequently increases the affinity of the enzyme for the substrate L-cysteine. Mass spectrometry and mutagenesis studies further established that the residue Cys255 is crucial for oxidation sensing. Oxidative stress-mediated sulfenylation of Cys255 leads to a sulfenic acid intermediate that spontaneously forms an intramolecular disulfide bond with the vicinal thiol group of Cys252. Moreover, we demonstrate that exogenous hydrogen peroxide (H₂O₂) and endogenous H₂O₂ triggered by vascular endothelial growth factor (VEGF) promote cellular H₂S production through the enhancement of CSE activity under oxidative stress conditions. By contrast, incubation with H₂O₂ or VEGF did not significantly enhance cellular H₂S production in the presence of PEG-catalase, an enzymatic cell-permeable H₂O₂ scavenger with high H₂O₂ specificity. Taken together, we report a new posttranslational modification of CSE that provides a molecular mechanism for H₂O₂/H₂S crosstalk in cells under oxidative stress.

Cystathionine β-synthase (CBS) overexpression is related to the proliferation and migration of human colon cancers. Targeted therapy that inhibits CBS has achieved promising effects in colon cancer treatments, but no selective inhibitor of CBS is available. In our previous study, a natural biflavonoid compound, sikokianin C, was identified as a potent and selective inhibitor of CBS. However, the mode of action of this compound and its antitumor efficacy in vivo remain unknown. In the present study, we have demonstrated that sikokianin C selectively inhibits CBS activity in a competitive manner, and the five key residues involved in the binding of sikokianin C to the substrate channel of CBS protein were identified via a combination of molecular docking and site-directed mutagenesis. Additionally, we analyzed the antitumor efficacy of sikokianin C against human colon cancer cells in vitro and in vivo. Sikokianin C greatly suppressed the proliferation of HT29 colon cancer cells with an IC₅₀ value of 1.6 μM, and CBS is the target of sikokianin C in mammalian cells, as evidenced by CBS knockdown analyses. Moreover, sikokianin C induced the apoptosis of HT29 cancer cells in a dose dependent manner. Treating mice with sikokianin C dramatically reduced the tumor volume and the weight of the colon cancer xenograft in vivo. These results indicate that the selective CBS inhibitor sikokianin C can potentially be used for the treatment of colon cancer.

S-adenosylmethionine (SAM), an important metabolite in all living organisms, has been widely used to treat various diseases. To develop a simple and efficient method to produce SAM, an engineered variant of the methionine adenosyltransferase (MAT) from Escherichia coli was investigated for its potential use in the enzymatic synthesis of SAM due to its significantly decreased product inhibition. The recombinant I303V MAT variant was successfully produced at a high level (~800 mg/L) with approximately four-fold higher specific activity than the wild-type MAT. The recombinant I303V MAT was covalently immobilized onto the amino resin and epoxy resin in order to obtain a robust biocatalyst to be used in industrial bioreactors. The immobilized preparation using amino resin exhibited the highest activity coupling yield (~84%), compared with approximately 3% for epoxy resin. The immobilized enzyme was more stable than the soluble enzyme under the reactive conditions, with a half-life of 229.5 h at 37 °C. The Km^ATP value (0.18 mM) of the immobilized enzyme was ca. two-fold lower than that of the soluble enzyme. Furthermore, the immobilized enzyme showed high operational stability during 10 consecutive 8 h batches, with the substrate adenosine triphosphate (ATP) conversion rate above 95% on the 50-mM scale.