Transsulfuration in Saccharomyces cerevisiae is not dependent on heme: purification and characterization of recombinant yeast cystathionine beta-synthase.
Cystathionine beta-synthase [CBS; L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration in both yeast and humans. It has been established previously that human CBS is a hemeprotein but although the heme group appears to be essential for CBS activity, the exact function of the heme group is unknown. CBS activity is absent in heme deficient strains of Saccharomyces cerevisiae grown without heme supplementation. CBS activity can be restored by supplementing these strains with heme, implying that there is a heme requirement for yeast CBS. We subcloned, overexpressed and purified yeast CBS. The yeast enzyme shows absolute pyridoxal 5'-phosphate (PLP) dependence for activity but we could find no evidence for the presence of a heme group. Given the degree of sequence and mechanistic similarity between yeast and human CBS, this result indicates that heme is unlikely to play a direct catalytic role in the human CBS reaction mechanism. Further characterization revealed that, in contrast to human CBS, S-adenosylmethionine (AdoMet) does not activate yeast CBS. Yeast CBS was found to be coordinately regulated with proliferation in S. cerevisiae. This finding is the most likely explanation of the observed apparent heme dependence of transsulfuration in vivo.
Maclean
K N
KN
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
Janosík
M
M
Oliveriusová
J
J
Kery
V
V
Kraus
J P
JP
D000595:Amino Acid Sequence; D002384:Catalysis; D002455:Cell Division; D003001:Cloning, Molecular; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D011732:Pyridoxal Phosphate; D013455:Sulfur; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase