Active Cystathionine β-Synthase Can Be Expressed in Heme-free Systems in the Presence of Metal-substituted Porphyrins or a Chemical Chaperone

Abstract

Cystathionine ␤-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30 -60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methionine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine.

Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones

“…Our previous results indicated that heme does not function in redox sensing, ligand binding, or catalysis but rather supported the structural role of heme in proper folding and/or subunit assembly (22,28,(32)(33)(34)(35). With respect to this hypothesis and the presented data, heme saturation could serve as a marker for properly folded CBS and could be indicative of the activity of purified CBS.…”

“…Moreover, these misfolded CBS aggregates were devoid of heme, which resulted in the hypothesis that heme may be necessary for correct folding and active tetramer formation of CBS. Our recent work suggested that despite the heme being crucial for proper CBS folding and assembly, it is not essential for expression of active CBS and can be substituted with other metalloporphyrins or even completely replaced by including a chemical chaperone such as TMAO in the growth medium of a heme-deficient S. cerevisiae (28). The usefulness of chemical chaperones in restoration of mutant CBS function was first demonstrated by Singh et al (16).…”

S-Glutathionylation Enhances Human Cystathionine β-Synthase Activity Under Oxidative Stress Conditions

“…residues in CBS by peroxynitrite results in alterations in the heme pocket, leading to loss of cysteinate coordination by the heme ligand Cys52 and concomitant inactivation of CBS (5). While a structural role for the heme in stabilizing the CBS structure has been invoked (29), homologous CBSs from lower organisms, for example, yeast, are stable without this cofactor (18). The central catalytic domain of CBS contains the PLP cofactor essential for catalysis and a CXXC motif, whose function, if any, has yet to be determined (30,48).…”

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).…”