Cystathionine-β-synthase, also known asCBS, is anenzyme (EC4.2.1.22) that in humans is encoded by theCBSgene. It catalyzes the first step of thetranssulfuration pathway, fromhomocysteine tocystathionine:[5]
CBS uses thecofactorpyridoxal-phosphate (PLP) and can beallosterically regulated by effectors such as the ubiquitous cofactorS-adenosyl-L-methionine (adoMet). This enzyme belongs to the family oflyases, to be specific, the hydro-lyases, which cleave carbon-oxygen bonds.
CBS is a multidomain enzyme composed of an N-terminal enzymatic domain and twoCBS domains. TheCBS gene is the most common locus for mutations associated withhomocystinuria.[6]
Thesystematic name of this enzyme class isL-serine hydro-lyase (adding homocysteine;L-cystathionine-forming). Other names in common use include:
Methylcysteine synthase was assigned theEC number EC 4.2.1.23 in 1961. A side-reaction of CBS caused this. The EC number EC 4.2.1.23 was deleted in 1972.[7]
The human enzyme cystathionine β-synthase is atetramer and comprises 551amino acids with a subunit molecular weight of 61 kDa. It displays a modular organization of three modules with the N-terminal heme domain followed by a core that contains thePLP cofactor.[9] The cofactor is deep in the heme domain and is linked by a Schiff base.[10] ASchiff base is afunctional group containing a C=N bond with the nitrogen atom connected to anaryl oralkyl group. The heme domain is composed of 70 amino acids and it appears that the heme only exists inmammalian CBS and is absent in yeast andprotozoan CBS. At the C-terminus, the regulatory domain of CBS contains a tandem repeat of two CBS domains of β-α-β-β-α, a secondary structure motif found in other proteins.[9] CBS has a C-terminal inhibitory domain. The C-terminal domain of cystathionine β-synthase regulates its activity via both intrasteric and allosteric effects and is important for maintaining the tetrameric state of the protein.[9] This inhibition is alleviated by binding of theallosteric effector,adoMet, or by deletion of the regulatory domain; however, the magnitude of the effects differ.[9] Mutations in this domain are correlated withhereditary diseases.[11]
The heme domain contains an N-terminal loop that binds heme and provides the axialligands C52 and H65. The distance of heme from thePLP binding site suggests its non-role in catalysis, however deletion of the heme domain causes loss ofredox sensitivity, therefore it is hypothesized that heme is a redox sensor.[10] The presence of protoporphyrin IX in CBS is a unique PLP-dependent enzyme and is only found in the mammalian CBS.D. melanogaster andD. discoides have truncatedN-terminal extensions and therefore prevent the conservedhistidine andcysteine heme ligandresidues. However, theAnopheles gambiae sequence has a longer N-terminal extension than the human enzyme and contains the conservedhistidine andcysteineheme ligand residues like the humanheme. Therefore, it is possible that CBS in slime molds and insects are hemeproteins that suggest that theheme domain is an early evolutionary innovation that arose before the separation of animals and the slime molds.[9] ThePLP is an internalaldimine and forms aSchiff base with K119 in the active site. Between the catalytic and regulatory domains exists a hypersensitive site that causes proteolytic cleavage and produces a truncateddimeric enzyme that is more active than the original enzyme. Both truncated enzyme and the enzyme found in yeast are not regulated by adoMet. The yeast enzyme is also activated by the deletion of the C-terminal to produce the dimeric enzyme.[9]
As of late 2007, twostructures have been solved for this class of enzymes, withPDB accession codes1JBQ and1M54.
| cystathionine beta-synthase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 Cystathionine beta synthase homodimer, Human | |||||||||
| Identifiers | |||||||||
| EC no. | 4.2.1.22 | ||||||||
| CAS no. | 9023-99-8 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| Gene Ontology | AmiGO /QuickGO | ||||||||
| |||||||||
Transsulfuration, catalyzed by CBS, convertshomocysteine tocystathionine, which cystathione gamma lyase converts tocysteine.[12]
CBS occupies a pivotal position in mammalian sulfur metabolism at thehomocysteine junction where the decision to conservemethionine or to convert it to cysteine via thetranssulfuration pathway, is made. Moreover, the transsulfuration pathway is the only pathway capable of removing sulfur-containingamino acids under conditions of excess.[9]
In analogy with other β-replacement enzymes, the reaction catalyzed by CBS is predicted to involve a series ofadoMet-bound intermediates. Addition ofserine results in atranschiffization reaction, which forms of an externalaldimine. Thealdimine undergoes proton abstraction at the α-carbon followed by elimination to generate an amino-acrylate intermediate. Nucleophilic attack by the thiolate of homocysteine on the aminoacrylate and reprotonation at Cα generate the external aldimine ofcystathionine. A finaltransaldimination reaction releases the final product, cystathionine.[9] The final product,L-cystathionine can also form an aminoacrylate intermediate, indicating that the entire reaction of CBS is reversible.[13]
The measured V0 of an enzyme-catalyzed reaction, in general, reflects the steady state (where [ES] is constant), even though V0 is limited to the early part of a reaction, and analysis of these initial rates is referred to as steady-state kinetics. Steady-state kinetic analysis of yeast CBS yields parallel lines. These results agree with the proposed ping-pong mechanism in which serine binding and release of water are followed by homocysteine binding and release of cystathionine. In contrast, the steady-stateenzyme kinetics of rat CBS yields intersecting lines, indicating that the β-substituent of serine is not released from the enzyme prior to binding of homocysteine.[9]
One of the alternate reactions involving CBS is the condensation ofcysteine with homocysteine to form cystathionine andhydrogen sulfide (H2S).[13] H2S in the brain is produced fromL-cysteine by CBS. This alternative metabolic pathway is also dependent onadoMet.[14]
CBS enzyme activity is not found in all tissues and cells. It is absent from heart, lung, testes, adrenal, and spleen in rats. In humans, it has been shown to be absent in heart muscle and primary cultures of human aorticendothelial cells. The lack of CBS in these tissues implies that these tissues are unable to synthesize cysteine and that cysteine must be supplied from extracellular sources. It also suggests that these tissues might have increased sensitivity to homocysteine toxicity because they cannot catabolize excess homocysteine via transsulfuration.[13]
Allosteric activation of CBS byadoMet determines the metabolic fate ofhomocysteine. Mammalian CBS is activated 2.5-5-fold by AdoMet with adissociation constant of 15 μM.[6]AdoMet is an allosteric activator that increases theVmax of the CBS reaction but does not affect theKm for the substrates. In other words, AdoMet stimulates CBS activity by increasing the turnover rate rather than the binding of substrates to the enzyme.[9] This protein may use themorpheein model ofallosteric regulation.[15]
Human CBS performs a crucial step in thebiosynthetic pathway of cysteine by providing a regulatory control point for AdoMet. Homocysteine, after being methylated tomethionine, can be converted to AdoMet, which donatesmethyl groups to a variety of substrates, e.g.,neurotransmitters,proteins, andnucleic acids. AdoMet functions as an allosteric activator of CBS and exerts control on its biosynthesis: low concentrations of AdoMet result in low CBS activity, thereby funneling homocysteine into thetransmethylation cycle toward AdoMet formation. In contrast, high adoMet concentrations funnel homocysteine into thetranssulfuration pathway towardcysteine biosynthesis.[16]
In mammals, CBS is a highly regulated enzyme, which contains aheme cofactor that functions as a redox sensor,[11] that can modulate its activity in response to changes in the redox potential. If the resting form of CBS in the cell hasferrous (Fe2+) heme, the potential exists for activating the enzyme under oxidizing conditions by conversion to theferric (Fe3+) state.[9] The Fe2+ form of the enzyme is inhibited upon binding CO or nitric oxide, whereas enzyme activity is doubled when the Fe2+ is oxidized to Fe3+. The redox state of theheme is pH dependent, with oxidation of Fe2+–CBS to Fe3+–CBS being favored at low pH conditions.[17]
Since mammalian CBS contains a heme cofactor, whereas yeast and protozoan enzyme fromTrypanosoma cruzi do not have heme cofactors, researchers have speculated that heme is not required for CBS activity.[9]
CBS is regulated at the transcriptional level byNF-Y,SP-1, andSP-3. In addition it is upregulated transcriptionally byglucocorticoids andglycogen, and downregulated byinsulin. Methionine upregulates CBS at the post-transcriptional level.
Down syndrome is a medical condition characterized by an overexpression of cystathionine beta synthase (CBS) and a low level of homocysteine in the blood.It has been speculated that cystathionine beta synthase overexpression could be the major culprit in this disease (along with dysfunctioning of GabaA and Dyrk1a). The phenotype of Down syndrome is the opposite of hyperhomocysteinemia (described below). Pharmacologicals inhibitors of CBS have been patented by the Jerome Lejeune Foundation (November 2011) and trials (animals and humans are planned).
Hyperhomocysteinemia is a medical condition characterized by an abnormally large level ofhomocysteine in the blood. Mutations in CBS are the single most common cause of hereditary hyperhomocysteinemia. Genetic defects that affect theMTHFR,MTR, andMTRR/MS enzyme pathways can also contribute to high homocysteine levels. Inborn errors in CBS result in hyperhomocysteinemia with complications in the cardiovascular system leading to early and aggressive arterial disease. Hyperhomocysteinemia also affects three other major organ systems including the ocular, central nervous, and skeletal.[9]
Homocystinuria due to CBS deficiency is a special type of hyperhomocysteinemia. It is a rare, hereditary recessive autosomal disease, in general diagnosed during childhood. A total of 131 different homocystinuria-causing mutations have been identified. A common functional feature of the mutations in the CBS domains is that the mutations abolish or strongly reduce activation byadoMet.[16] No specific cure has been discovered for homocystinuria; however, many people are treated using high doses ofvitamin B6, which is a cofactor of CBS.
Cystathionine beta synthase (CBS) is involved inoocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary and research is now focused on determining the location and expression during follicle development in the ovaries.[18]
Absence of Cystathionine beta synthase in mice provokes infertility due to the loss of uterine protein expression.[19]
The genes that control CBS enzyme expression may not operate at 100% efficiency in individuals who have one of the SNPs (single-nucleotide polymorphisms, a type ofmutations) that affect this gene. Known variants include the A360A, C699T, I278T, N212N, and T42N SNPs (among others). These SNPs, which have varied effects on the effectiveness of the enzyme, can be detected with standard DNA testing methods.
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