Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii

Abstract

Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T. gondii CGL gene encodes a functional enzyme. Herein, we cloned and biochemically characterized cDNA encoding CBS from T. gondii (TgCBS), which represents a first example of protozoan CBS that does not bind heme but possesses two C-terminal CBS domains. We demonstrated that TgCBS can use both serine and O-acetylserine to produce cystathionine, converting these substrates to an aminoacrylate intermediate as part of a PLP-catalyzed β-replacement reaction. Besides a role in cysteine biosynthesis, TgCBS can also efficiently produce hydrogen sulfide, preferentially via condensation of cysteine and homocysteine. Unlike the human counterpart and similar to CBS enzymes from lower organisms, the TgCBS activity is not stimulated by S-adenosylmethionine. This study establishes the presence of an intact functional reverse transsulfuration pathway in T. gondii and demonstrates the crucial role of TgCBS in biogenesis of H 2 S.

Structure of Cystathionine β-Synthase from Toxoplasma gondii, a key enzyme in its H2S production machinery

“…2A). Through absorbance and circular dichroism (CD) studies, we ascertained that the mutant displayed no substantial differences compared to the wild type protein (24) in either the absorbance spectroscopic features (Fig. 2B) or protein conformation (secondary structure elements) ( Fig.…”

“…Similarly to the yeast enzyme (42), TgCBS belongs to the structural class C (Fig. 1), and does not respond to AdoMet stimulation (24). Herein we report the 3.2Å resolution crystal structure of an optimized protein construct (TgCBSΔ466-491) that contains the full-length TgCBS and only lacks amino acid residues 466-491, which belong to the long flexible loop linking the last two β-strands of the CBS2 motif.…”

“…A rare variant of this particular CBS, class B, is found in Caenorhabditis elegans (CeCBS), and includes two catalytic domains in tandem, of which only one binds PLP (19), (22). The next level of complexity, class C, appears in yeast (23) and some apicomplexa (24), whose CBSs conserve the catalytic domain, and incorporate an additional Cterminal Bateman module (25), (26). The Bateman module is in turn formed by two consecutive cystathionine β-synthase motifs (CBS1, CBS2), and its actual function in these organisms remains unknown.…”

“…We recently demonstrated that both CBS and CGL from Toxoplasma gondii (TgCBS and TgCGL) are functional in toxoplasma (24), (40), implying that the reverse transsulfuration pathway is operative in the parasite. In particular, TgCBS can use both Ser and O-acetylserine (OAS) to produce Cth (24). This catalytic ability links TgCBS to other enzymes such as OCBSs (41) and OASS, which also employ OAS as a substrate.…”

“…This catalytic ability links TgCBS to other enzymes such as OCBSs (41) and OASS, which also employ OAS as a substrate. Besides a role in cysteine biosynthesis, TgCBS is also strongly implicated in the production of H2S, being highly efficient in catalyzing the β-replacement of Cys with HCys (24). Similarly to the yeast enzyme (42), TgCBS belongs to the structural class C (Fig.…”