Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Abstract

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.

Oligomeric status of human cystathionine beta‐synthase modulates AdoMet binding

“…Figure 2 shows representative raw titrations and binding isotherms, while Table 1 summarizes thermodynamic parameters for AdoMet binding as determined by ITC. As previously reported [6,9,14], AdoMet binding to CBS WT is consistent with a binding capacity of about 1.5 moles of AdoMet/monomer into two types of independent binding sites. Attempts to analyze these binding isotherms using a sequential binding model with a total binding capacity of four binding sites per tetramer did not provide convergent fittings (data not shown), supporting the notion that these isotherms cannot be satisfactorily explained by four dependent (i.e.…”

“…Figure shows representative thermal denaturation profiles, while Table summarizes key denaturation parameters for the regulatory (RD) and the catalytic domains (CD) obtained for CBS WT and CBSΔ516‐525 variants using a two‐state irreversible denaturation model . As previously reported , thermal denaturation profile of CBS WT showed two main transitions with T m values of 51.2 °C and 67.2 °C corresponding to denaturation of RD and CD, respectively. The CBSΔ516‐525 variants presented similarly well resolved two thermal transitions to CBS WT, which T m for both RD and CD denaturation were generally up‐shifted by 2 °C.…”

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S -adenosylmethionine

“…Our hCBSΔ516-525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23,24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16,19,(25)(26)(27).…”

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

“…We now know that the enzyme responsible for this effect is cystathionine-␤synthase (CBS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, expressed in various cells of the liver, kidney and the nervous system, where it plays a role in cysteine biosynthesis and degradation [2][3][4][5][6]. CBS is unique among the PLP-dependent enzymes, as it also carries a N-terminal heme prosthetic group coordinated by Cys52 and His65 residues [7,8]. Although heme is not required for enzymatic activity, its presence can affect its catalytic activity serving as a redox sensor.…”

“…Although heme is not required for enzymatic activity, its presence can affect its catalytic activity serving as a redox sensor. The C-terminal region of CBS exerts an autoinhibitory function by partially shielding the active site that can be alleviated by S-adenosyl methionine binding [7,8].…”