Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase

Article abstract of DOI:10.1089/ars.2017.7009

Aims: The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H₂S). In this study, we assessed the role of CBS in H₂S biogenesis. Results: We show that despite discouraging enzyme kinetics of alternative H₂S-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of H₂S. The leading H₂S-producing reaction is condensation of cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine is more abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the main determinant of CBS H₂S productivity. Abundance of cysteine over serine, for example, in plasma, allowed for up to 43% of CBS activity being responsible for H₂S production, while excess of serine typical for intracellular levels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine and cysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H₂S pool. Innovation: Our study characterizes the H₂S-producing potential of CBS under biologically relevant conditions and highlights the serine-to-cysteine ratio as the main determinant of H₂S production by CBS in vivo. Conclusion: Our data clarify the function of CBS in H₂S biogenesis and the role of thioethers as surrogate H₂S markers.

Full text of DOI:10.1089/ars.2017.7009

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Antioxidants and Redox Signaling
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Page 1 of 47
DOI: 10.1089/ars.2017.7009
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ORIGINAL RESEARCH COMMUNICATION
Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine
Beta-Synthase
Tomas Majtan1*, Jakub Krijt2, Jitka Sokolová2, Michaela Křížková2, Maria A. Ralat3, Jana
Kent1, Jesse F. Gregory III3, Viktor Kožich2 and Jan P. Kraus1*
Short title: CBS function in H2S biogenesis
1Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA
2Institute of Inherited Metabolic Disorders, Charles University-First Faculty of Medicine
and General University Hospital in Prague, Prague, Czech Republic
3Food Science and Human Nutrition Department, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL, USA
*To whom the correspondence should be addressed:
Jan P. Kraus, University of Colorado, School of Medicine, 12800 E 19th Ave, Mail Stop 8313,
Aurora, CO, 80045, USA; tel: 303-724-3812, fax: 303-724-3838, e-mail:
jan.kraus@ucdenver.edu
Tomas Majtan, University of Colorado, School of Medicine, 12800 E 19th Ave, Mail Stop
8313, Aurora, CO, 80045, USA; tel: 303-724-3813, fax: 303-724-3838, e-mail:
tomas.majtan@ucdenver.edu

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Word count (excluding references & figure legends): 7,021; Number of references: 62;
Number of greyscale illustrations: 6; Number of color illustrations: none
Keywords: enzyme kinetics, alternative reactions, homocystinuria, animal model, sulfur
metabolism

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ABSTRACT
AIMS: The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and
cystathionine gamma lyase (CGL) are thought to be the major source of hydrogen sulfide
(H2S). Here, we assessed the role of CBS in H2S biogenesis.
RESULTS: We show that despite discouraging enzyme kinetics of alternative H2S-producing
reactions utilizing cysteine compared to the canonical condensation of serine and
homocysteine, our simulations of substrate competitions at biologically relevant
conditions suggest that cysteine is able to partially compete with serine on CBS, thus
leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is
a condensation of cysteine with homocysteine, while cysteine desulfuration plays
dominant role when cysteine is more abundant than serine and homocysteine is limited.
We found that serine to cysteine ratio is the main determinant of CBS H2S productivity.
Abundance of cysteine over serine in e.g. plasma allowed for up to 43% of CBS activity
being responsible for H2S production, while excess of serine typical for intracellular levels
effectively limited such activity to less than 1.5%. CBS also produced lanthionine from
serine and cysteine and a third of lanthionine coming from condensation of two cysteines
contributed to H2S pool.
INNOVATION: Our study characterizes the H2S-producing potential of CBS under
biologically relevant conditions and highlights serine to cysteine ratio as the main
determinant of H2S production by CBS in vivo.
CONCLUSION: Our data clarifies the function of CBS in H2S biogenesis and the role of
thioethers as surrogate H2S markers.

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INTRODUCTION
Cystathionine beta-synthase (CBS) (EC 4.2.1.22) plays a crucial role in cysteine
biosynthesis from the essential amino acid methionine via cystathionine (Cth) in the
transsulfuration pathway (35). Cystathionine is generated by the CBS-catalyzed
condensation of serine (Ser) and homocysteine (Hcy), and is then cleaved by cystathionine
gamma-lyase (CGL) to yield cysteine (Cys). CBS is a member of the large -family of
pyridoxal-5’-phosphate (PLP)-dependent enzymes and its catalytic core has a high degree
of sequence and structural conservation with cysteine synthases and O-
acetylserine(thiol)lyases, the CBS orthologs in prokaryotes, plants and nematodes
(17,39,52,57). CBS catalyzes primarily -replacement reactions (Figure 1) (4,38). Despite
catalyzing mostly one reaction type, CBS can utilize a wide range of substrates. The
substrate specificity of CBS was originally investigated by Braunstein et al. (5). They
demonstrated that in addition to cystathionine formation from serine and homocysteine
(canonical activity), partially purified rat liver CBS catalyzed the production of thioethers
from serine, substituted analogues of serine, cysteine and S-alkyl derivatives of cysteine
upon their incubation with a variety of sulfhydryl compounds, such as β-mercaptoethanol
and cysteamine (Figure 1). They and others showed that CBS is also capable of H2S
production in vitro by both cysteine desulfuration (β-elimination reaction) and its
condensation with a number of other sulfhydryl compounds (β-replacement reactions)
(7,42,58,59).
H2S used to be considered a toxic gas until the discovery that it regulates a number
of physiological processes (16). Moreover, H2S has been implicated in various diseases
ranging from hypertension, atherosclerosis through lipid metabolism, diabetes,
inflammation to neuromodulation (53,60). Despite its importance, there is still no
consensus on the physiological levels of H2S in blood and/or tissues or on the most reliable
technique for measuring H2S in biological samples. On the contrary to the initial studies
reporting of up to 300 µM H2S in circulation (37), the current conservative estimate of the
physiological H2S levels in circulation is in the higher nanomolar to lower micromolar range