Human Cystathionine β-Synthase Is a Heme Sensor Protein. Evidence That the Redox Sensor Is Heme and Not the Vicinal Cysteines in the CXXC Motif Seen in the Crystal Structure of the Truncated Enzyme^,

Abstract

Elevated levels of homocysteine, a sulfur-containing amino acid, are correlated with increased risk for cardiovascular diseases and Alzheimers disease and with neural tube defects. The only route for the catabolic removal of homocysteine in mammals begins with the pyridoxal phosphate- (PLP-) dependent beta-replacement reaction catalyzed by cystathionine beta-synthase. The enzyme has a b-type heme with unusual spectroscopic properties but as yet unknown function. The human enzyme has a modular organization and can be cleaved into an N-terminal catalytic core, which retains both the heme and PLP-binding sites and is highly active, and a C-terminal regulatory domain, where the allosteric activator S-adenosylmethionine is presumed to bind. Studies with the isolated recombinant enzyme and in transformed human liver cells indicate that the enzyme is approximately 2-fold more active under oxidizing conditions. In addition to heme, the enzyme contains a CXXC oxidoreductase motif that could, in principle, be involved in redox sensing. In this study, we have examined the role of heme versus the vicinal thiols in modulating the redox responsiveness of the enzyme. Deletion of the heme domain leads to loss of redox sensitivity. In contrast, substitution of either cysteine with a non-redox-active amino acid does not affect the responsiveness of the enzyme to reductants. We also report the crystal structure of the catalytic core of the enzyme in which the vicinal cysteines are reduced without any discernible differences in the remainder of the protein. The structure of the catalytic core is compared to those of other members of the fold II family of PLP-dependent enzymes and provides insights into active site residues that may be important in interacting with the substrates and intermediates.

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

“…Whereas the heme domain (residues 1-45) lacks secondary structure, the PLP domain (residues 46-350) exhibits a previously described fold (13) containing two subdomains (A and B) that bind PLP at the interface. The N-terminal module of dCBS is very similar to the corresponding module of hCBS (11,12) (Fig S1). The long linker (α L , residues 351-377) sandwiched between the N-and C-terminal modules includes a helix and contacts residues from both modules.…”

“…Changes in the heme coordination environment are transmitted to the PLP site some 20 Å away and lead to inhibition of CBS activity via a shift from the ketoenamine to the enolimine tautomer of the PLP (10). Although the crystal structure of the catalytic core of hCBS is known (11,12), the architecture of the full-length protein is not. In this study, we report the structures of full-length Drosophila CBS (dCBS) in the substrate-free form (at 1.80-Å resolution) and in which two reaction intermediates, a carbanion (1.70 Å) and an aminoacrylate species (1.55 Å), have been captured (Table 1).…”

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

“…The crystal structure of the truncated CBS (1-413) has been since solved [30,31] and equimolar binding of heme to CBS subunits confirmed.…”

Structural basis of regulation and oligomerization of human cystathionine β-synthase, the central enzyme of transsulfuration

“…The N-terminal domain binds the heme cofactor, which is axially coordinated by C52 and H65. The role of heme in CBS is not fully understood, and both structural and regulatory functions have been attributed to it (10)(11)(12)(13). However, CBS from lower eukaryotes, such as yeast, lacks heme entirely, thus clearly indicating that is not essential for catalytic activity (14,15).…”

“…Recently, we identified two sets of AdoMetbinding sites with different structural and energetic features; these sites are capable of accommodating up to six ligands in the regulatory domain of the tetrameric full-length enzyme (19). The C-terminal truncation is accompanied by a change in the oligomeric status from a homotetramer to a homodimer (17), whose crystal structure was solved more than a decade ago (10,20). In 2010, Koutmos et al (21) presented the crystal structure of a fulllength CBS from Drosophila melanogaster (dCBS).…”