Current Structural Knowledge on Cystathionine β‐Synthase, a Pivotal Enzyme in the Transsulfuration Pathway


Cystathionine β‐synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyses the pyridoxal‐5′‐phosphate (PLP)‐dependent β‐replacement reaction that condenses l‐serine with L‐homocysteine to yield cystathionine and water. Besides this canonical reaction and using cysteine and homocysteine as substrates, CBS can also efficiently produce hydrogen sulfide (H2S) through alternative β‐replacement and β‐elimination processes. The structural information on the full‐length enzyme has remained elusive for decades and is still very scarce, but some advances in the recent years have uncovered its peculiar modular architecture, provided a glimpse of its conformational landscape and revealed some of the reaction intermediates formed during the catalysis. All these data have helped comprehend, at least partially, the regulatory mechanisms and the catalytic abilities of the enzyme across different organisms. This article aims to overview the current information on the CBS structure from its most sophisticated variants found in mammals to its simplest homologs in bacteria. A more detailed understanding of CBS structure and function is needed, which could subsequently serve as a basis for the development of drugs to treat human diseases, such as CBS‐deficient homocystinuria, Alzheimer diseases and some cancers, as well as of new antibiotics against multidrug‐resistant pathogenic bacteria.

Key Concepts

  • Reverse transsulfuration is a two‐step metabolic route that allows the conversion of the essential amino acid methionine into cysteine. This process generates hydrogen sulfide and impedes the accumulation of the toxic intermediate homocysteine.
  • Hydrogen sulfide is an important gasotransmitter involved in physiological functions such as neuroprotection and the regulation of blood pressure.
  • Homocystinuria consists of the abnormal accumulation of homocysteine, and is an inherited disorder due to the deficient activity of CBS. This pathology causes vascular thromboses, skeletal defects, mental retardation, and even early death.
  • The three‐dimensional structure of CBS provides a suitable template to develop drugs to treat homocystinuria and related pathologies in humans.
  • The comparative structural analysis of the CBS enzymes from different organisms are key to intervene in their sulfur metabolism, and thus represents a potential therapeutic approach against pathogens.

Keywords: cystathionine β‐synthase; transsulfuration; homocysteine; H2S; homocystinuria; S‐adenosyl‐l‐methionine; pyridoxal‐5′‐phosphate; crystallography

Figure 1. Metabolic Map of Transsulfuration. (a) The reverse transsulfuration route is linked to the folates and methionine cycles, which in turn are coupled to the sarcosine pathway. In mammals, reverse transsulfuration is very active in the liver and represents the sole source of cysteine, which is further converted into glutathione (GSH) by the action of glutamate–cysteine ligase (GCL) and glutathione synthase (GSS). Alternatively, homocysteine (Hcy) can be remethylated through the folates and methionine cycles generating methionine (Met). This process is catalysed by two enzymes: (1) betaine homocysteine methyltransferase (BHMT), a liver‐ and kidney‐specific enzyme that converts Hcy into Met using betaine as co‐substrate; (2) methionine synthase (MS), which uses 5‐methyltetrahydrofolate (5‐MTHF) as methyl donor and generates tetrahydrofolate (THF), which in turn is converted to 5,10‐methylenetetrahydrofolate (5,10‐MTHF) by the enzyme 5,10‐methylenetetrahydrofolate synthetase (MTHFS) and regenerated to 5‐MTHF by methyltetrahydrofolate reductase (MTHFR). (b) Some organisms, like bacteria, are able to perform a forward transsulfuration pathway and a de‐novo cysteine synthesis, which allow the conversion of Cys into Hcy, and the synthesis of Cys from Ser, respectively. However, mammals possess only the reverse transsulfuration pathway, thus making Met an essential amino acid, which can be converted into Cys. The enzymes involved in these processes are cystathionine γ‐synthase (CGS), cystathionine β‐lyase (CBL), cysteine synthase (CS) and serine acetyltransferase (SAT). Abbreviations: (Met, methionine; MAT, methionine S‐adenosyltransferase; AdoMet, S‐adenosyl‐l‐methionine; GNMT, glycine N‐methyltransferase; AdoHcy, S‐adenosylhomocysteine; SAHH, S‐adenosylhomocysteine hydrolase; CBS, cystathionine β‐synthase; CGL (or CSE, CTH), cystathionine γ‐lyase; GCLC/M, glutamate‐cysteine ligase, catalytic/modulator subunit; BHMT, betaine homocysteine methyltransferase; MS, methionine synthase; 5‐MTHF, 5‐methyltetrahydrofolate; THF, tetrahydrofolate; MTHFS, 5,10‐methyltetrahydrofolate synthase; 5,10‐dimethyltetrahydrofolate; DHF, dihydrofolate; DMG, dimethylglycine; SDH, sarcosine dehydrogenase.
Figure 10. AdoMet‐binding site of HsCBS. Sticks representation of residues involved in AdoMet binding at site S2 of the Bateman module.
Figure 2. Reactions catalyzed by enzymes of reverse transsulfuration. CBS is cystathionine β‐synthase; CGL is cystathionine γ‐lyase. The square frames highlight the canonical processes. H2S is hydrogen sulfide.
Figure 3. Domain distribution of CBS in different organisms. (top) Five different architectures are known for CBSs: (a) Mammalian CBS enzymes (e.g. Homo sapiens HsCBS, Mus musculus MmCBS, Rattus norvegicus RnCBS) contain the N‐terminal heme‐binding domain (red), a central catalytic domain that hosts PLP (yellow) and the C‐terminal Bateman regulatory domain (blue) that binds the allosteric activator AdoMet (magenta). The CBS2 motif contains a long loop that participates in protein tetramerisation. The protein presents two conformations: AdoMet‐free basal (low activity) and AdoMet‐bound activated (high activity). (b) CBS from fruit fly (Drosophila melanogaster, DmCBS) or honeybee (Apis mellifera, AmCBS) contains the same domain architecture found in mammals but is not regulated by AdoMet. The length of the loop of the CBS2 domain is short. The enzyme adopts a unique activated conformation and forms dimers. (c) CBS from yeast (Saccharomyces cerevisiae ScCBS) lacks the heme‐binding domain and is not allosterically regulated by AdoMet. The loop of the CBS2 motif is similarly long as in mammals and the enzyme forms tetramers. (d) CBS enzymes from bacteria (e.g. Bacillus anthracis BaCBS, Lactobacillus plantarum LpCBS) and protozoa (e.g. Trypanosoma cruzi TcCBS) are dimeric and lacks the Bateman module. The protein presents a unique conformation that is active. (e) CBS from roundworm Caenorhabditis elegans (CeCBS) contains two linked catalytic domains, of which only one hosts the PLP cofactor and thus is active. Lower part depicts conformations found in CBS enzymes, for which the crystal structures have been determined (middle, left) Basal conformation of HsCBSΔ516‐525 (engineered dimeric construct) (middle, right) AdoMet‐bound activated conformation of HsCBSΔ516‐525 E201S variant (dimeric construct). (bottom, left) Active conformation of bacterial CBS. (bottom, right) Constitutively active conformation of insect CBS. The heme domain, the catalytic domain and the Bateman module are coloured in red, yellow and blue, respectively. The interdomain linker is in green. AdoMet is in magenta. The two complementary subunits are represented in opaque and transparent surfaces, respectively. Note: The letters (a) to (e) indicate the five different architectures found in CBSs as outlined in the upper portion of the figure.
Figure 4. Human CBS. (a) Amino acid sequence of the catalytic core of HsCBS. Secondary elements are indicated. The heme‐binding domain is in red ribbons. Heme is in red sticks. (b) The catalytic domain (in ribbons) is split into two subdomains: static (in yellow) and mobile (in cyan). The linker between the catalytic core and the Bateman module is in green. (c) The C‐terminal regulatory Bateman module consists of two CBS motifs, CBS1, CBS2. Two main cavities, S1 and S2, are formed. S2 represents the AdoMet‐binding site.
Figure 5. Heme‐binding domain of HsCBS. (a) Ribbons representation of the heme‐binding domain (in red) inserted in the catalytic core, that also contains the catalytic domain (in grey). Helices α8 and α9 from the catalytic domain interact with heme (in sticks). (b) Amino acid residues forming the heme‐binding pocket or interacting with heme.
Figure 6. Residues connecting heme and PLP. (a) B1 and B2 are two consensus sequences involved in catalysis (see also Figure c). Based on Devi S, Tarique KF, Ali MF, Rehman SAA and Gourinath S. (2019) Identification and characterization of Helicobacter pylori O‐acetylserine‐dependent cystathionine β‐synthase, a distinct member of the PLP‐II family. Molecular Microbiology 112 (2): 718–739. (b) Ketoenamine and enolimine forms of PLP.
Figure 7. Conformations of HsCBS. Binding of AdoMet to the Bateman module triggers a conformational change that makes HsCBS transition from the basal towards the activated state. The colours of the domains follow the criteria adopted in Figure .
Figure 8. Catalytic domain of CBS. (a) The catalytic domain of HsCBS is formed by two subdomains: static (in yellow) and mobile (cyan). The catalytic cavity hosting PLP (represented as surface) is located between these two blocks. In the basal conformation, the regulatory Bateman module is placed above the mobile subdomain, which is compressed towards the cleft thus impairing the access of substrates to the PLP. (b) Superimposition of the catalytic domain in the basal (light grey) and activated (dark grey) states. The mobile subdomain (in cyan) shifts away from the entrance of the cavity in the activated state, allowing the access of substrates to the PLP. (c) Consensus sequences (blocks) B1‐5 involved in catalysis. Residues (in sticks) are coloured according to the block they belong to, following the criteria of panel b. Based on Devi S, Tarique KF, Ali MF, Rehman SAA and Gourinath S. () Identification and characterization of Helicobacter pylori O‐acetylserine‐dependent cystathionine β‐synthase, a distinct member of the PLP‐II family. Molecular Microbiology 112(2):718–739.
Figure 9. Conformational change triggered by AdoMet. (a) Binding of AdoMet (in sticks) to the site S2 cavity of the Bateman module triggers a conformational change consisting in a relative rotation of the two CBS motifs. Basal and activated conformations are coloured in light and dark blue, respectively. (b) The orientation of the interfacial helices of the Bateman module changes upon binding of AdoMet and that weakens their initial interactions with the catalytic core (in grey). (c) In the AdoMet‐bound form, the Bateman modules form a disc‐like structure known as ‘CBS module’.


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González‐Recio, Irene, Fernández‐Rodríguez, Carmen, Simón, Jorge, Goikoetxea‐Usandizaga, Naroa, Martínez‐Chantar, Maria Luz, Astegno, Alessandra, Majtan, Tomas, and Martinez‐Cruz, Luis Alfonso(Oct 2020) Current Structural Knowledge on Cystathionine β‐Synthase, a Pivotal Enzyme in the Transsulfuration Pathway. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0028966]