X-ray of coiled coils and PLP-dependent enzymes (P. Burkhard)

CBS Cystathionine beta Synthase

CBS is a unique heme enzyme that catalyzes a PLP-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an autosomal recessively inherited disease of sulfur metabolism.
A truncated form of CBS in which the COOH-terminal amino acid residues had been deleted has been prepared. The truncated CBS subunits form a dimer in contrast to the full-length enzyme, which forms tetramers and higher oligomers. The truncated CBS yield crystals diffracting to 2.6 Å which belong to the space group P3₁.

Schematic representation of the tertiary fold of a dimer of CBS. The central beta-sheets are colored in blue and the surrounding alpha-helices are colored in magenta. In ball and stick representation and marked by a black box are the active site PLP, the heme, and the oxidoreductase motif. The view is down the non-crystallographic two-fold axis which relates the two subunits of the dimer.

The active site region of CBS (grey) in a superposition with the active site of OASS (green). The sequences as well as the structure of the two enzymes are very similar. A superposition of the 25 structurally most conserved residues yields an rmsd of 1.6 Å of their Ca positions. The substrate analogue of OASS methionine indicates the probable binding mode of the first substrate serine and also the region where the second substrate homocysteine is expected to bind to.

The heme binding site of CBS with heme in green and the surrounding residues in grey. The two residues His65 and Cys52 are the ligands to the heme iron (dark red). The difference density for the cofactor is shown in red contoured at 3.5 sigma.

Using polarized absorption spectroscopy of CBS in the crystalline state the dissociation constant of L-serine was found to be 6 mM, close to that determined in solution. The accumulation of the alpha-aminoacrylate Schiff base was pH independent between 6 and 9 but its stability decreases at pH higher than 8. The alpha-aminoacrylate-enzyme crystals react with homocysteine to form cystathionine, indicating that CBS crystals catalyze both the beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by CBS.

Publications

Meier, M., Oliveriusova, J., Kraus, J.P., & Burkhard P. (2003). Structural insights into mutations of cystathionine beta-synthase. Biochim Biophys Acta., 1647(1-2):206-13. (MEDLINE)

Meier, M., Janosik, M., Kery, V., Kraus, J. & Burkhard, P. (2001). Structure of human cystathionine b-synthase: A unique pyridoxal 5'-phosphate dependent hemeprotein. EMBO Journal, 20 (15), 3910-3916. (MEDLINE).

Bruno, S., Schiaretti, F., Burkhard, P., Kraus, J.P., Janosik, M. & Mozzarelli, A. (2001). Functional properties of human cystathionine beta-synthase crystals. J Biol Chem, 276, 16-19. (MEDLINE)

Janosik, M., Meier, M., Oliveriusova, J., Burkhard, P. & Kraus, J. (2001). Crystallization and preliminary X-ray diffraction analysis of the active core of human recombinant cystathionine b-synthase: An enzyme involved in cardiovascular disease. Acta Cryst, D57, 289-291. (MEDLINE)

	Schematic representation of the tertiary fold of a dimer of CBS. The central beta-sheets are colored in blue and the surrounding alpha-helices are colored in magenta. In ball and stick representation and marked by a black box are the active site PLP, the heme, and the oxidoreductase motif. The view is down the non-crystallographic two-fold axis which relates the two subunits of the dimer.
	The active site region of CBS (grey) in a superposition with the active site of OASS (green). The sequences as well as the structure of the two enzymes are very similar. A superposition of the 25 structurally most conserved residues yields an rmsd of 1.6 Å of their Ca positions. The substrate analogue of OASS methionine indicates the probable binding mode of the first substrate serine and also the region where the second substrate homocysteine is expected to bind to.
	The heme binding site of CBS with heme in green and the surrounding residues in grey. The two residues His65 and Cys52 are the ligands to the heme iron (dark red). The difference density for the cofactor is shown in red contoured at 3.5 sigma.
	Using polarized absorption spectroscopy of CBS in the crystalline state the dissociation constant of L-serine was found to be 6 mM, close to that determined in solution. The accumulation of the alpha-aminoacrylate Schiff base was pH independent between 6 and 9 but its stability decreases at pH higher than 8. The alpha-aminoacrylate-enzyme crystals react with homocysteine to form cystathionine, indicating that CBS crystals catalyze both the beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by CBS.