Deletion mutagenesis of human cystathionine beta-synthase. Impact on activity, oligomeric status, and S-adenosylmethionine regulation.
Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.
Oliveriusová
Jana
J
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
Kery
Vladimír
V
Maclean
Kenneth N
KN
Kraus
Jan P
JP
D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry*; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D017384:Sequence Deletion
D001704:Biopolymers; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006418:Heme; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase