Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation

Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mut ations in the human enzyme. The mutations, which lead to accumulation of L- homocysteine, are linked to homocystinuria and cardiovascular diseases. Her e we characterize the domain architecture of the heme-independent yeast cys tathionine beta-synthase. Our finding that the homogeneous recombinant trun cated enzyme (residues 1-353) is catalytically active and binds pyridoxal p hosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain. Removal of the C-terminal residues 354-507 incr eases the specific activity and alters the steady-state kinetic parameters including the K-d for pyridoxal phosphate, suggesting that the C-terminal r esidues 354-507 compose a regulatory domain, The yeast enzyme, unlike the h uman enzyme, is not activated by S-adenosyl-L-methionine. The truncated yea st enzyme is a dimer, whereas the full-length enzyme is a mixture of tetram er and octamer, suggesting that the C-terminal domain plays a role in the i nteraction of the subunits to form higher oligomeric structures, The N-term inal catalytic domain is more stable and less prone to aggregate than full- length enzyme and is thus potentially more suitable for structure determina tion by X-ray crystallography. Comparisons of the yeast and human enzymes r eveal significant differences in catalytic and regulatory properties.