Insights into Domain Organization and Regulatory Mechanism of Cystathionine Beta-Synthase from Toxoplasma gondii

Abstract

Cystathionine beta-synthase (CBS) is a key regulator of homocysteine metabolism. Although eukaryotic CBS have a similar domain architecture with a catalytic core and a C-terminal Bateman module, their regulation varies widely across phyla. In human CBS (HsCBS), the C-terminus has an autoinhibitory effect by acting as a cap that avoids the entry of substrates into the catalytic site. The binding of the allosteric modulator AdoMet to this region alleviates this cap, allowing the protein to progress from a basal toward an activated state. The same activation is obtained by artificial removal or heat-denaturation of the Bateman module. Recently, we reported the crystal structure of CBS from Toxoplasma gondii (TgCBS) showing that the enzyme assembles into basket-like dimers similar to the basal conformers of HsCBS. These findings would suggest a similar lid function for the Bateman module which, as in HsCBS, should relax in the absence of the C-terminal module. However, herein we demonstrate that, in contrast with HsCBS, removal of the Bateman module in TgCBS through deletion mutagenesis, limited proteolysis, or thermal denaturation has no effects on its activity, oligomerization, and thermal stability. This opposite behavior we have now found in TgCBS provides evidence of a novel type of CBS regulation.

Structural basis of the inhibition of cystathionine γ‐lyase from Toxoplasma gondii by propargylglycine and cysteine

“…We recently demonstrated that T. gondii encodes both the CBS (TgCBS) and CGL (TgCGL) enzymes of reverse transsulfuration (Conter et al, 2020(Conter et al, , 2022Fern andez-Rodríguez et al, 2021;Maresi et al, 2018). TgCGL (UniProtKB A0A125YN40, 46 kDa) shares a high amino acid identity with its homologs from Trypanosomes and Leishmania parasites (58% homology) but is less similar to the human and yeast counterparts (40% and 37% for H. sapiens and S. cerevisiae, respectively).…”

Key substrate recognition residues in the active site of cystathionine beta‐synthase from Toxoplasma gondii

“…1,6,8,9,26,27 CBS from T. gondii provides a particularly valuable model for exploring the enzyme-substrate specificity because it similarly processes both L-Ser and L-OAS for β-replacement activity to produce L-Cth. [10][11][12] Our analysis of a representative group of 11 prokaryotic and eukaryotic CBSs and OCBSs, for which substrate preference is known, revealed that the polar residues Ser82, Tyr158, and Tyr248 (numbered according to the conventional yeast CBS) are conserved among canonical CBS sequences, while distinct hydrophobic residues (Ala, Phe, and Val) are usually found in OCBSs. Considering the proposal of Devi et al, this provides indirect support that these positions play critical roles in defining the L-Ser/L-OAS preference of the two proteins.…”

“…Absorption spectra were recorded on a Jasco V-750 spectrophotometer in a buffer containing 20 mM sodium phosphate pH 8.5 at 25 C using a protein concentration of 12 μM. 10,11 CD spectra were recorded on a Jasco J-1500 CD spectropolarimeter equipped with a Peltier-type temperature controller as previously described. 16,17 Briefly, near UV-Vis (250-600 nm) spectra of 1 mg/mL TgCBS variants were recorded in 1-cm path length quartz cuvette at a scan speed of 50 nm/min.…”

“…8 We recently showed that CBS from Toxoplasma gondii (TgCBS) can use both L-Ser and L-OAS to give L-Cth and is unresponsive to AdoMet, likely linking its catalytic ability to the OCBS enzymes. [10][11][12] However, TgCBS possesses higher sequence similarity to CBS than OCBS as it contains a C-terminal Bateman module and, with respect to the above-mentioned active site fingerprint, the residues of canonical CBS (Ser, Tyr, and Tyr). The particular structural features of TgCBS were recently unraveled from the crystal structure of the enzyme.…”

Structural Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma gondii

“…DSC experiments were conducted using a nano-DSC (TA instrument) with a cell volume of 300 μL. 58 Protein samples, containing 60–80 μM Cyp in 5 mM Hepes, pH 8.0; 10 mM NaCl; and 0.2% ethanol, in the presence or absence of CsA, were heated from 15 to 100 °C at a scan rate of 60 °C h –1 . Data were fitted according to a two-state model using NanoAnalyze software (TA instrument).…”