Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe

Abstract

D-serine is an endogenous coagonist for the N-methyl-D-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5'-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of L-serine to yield D-serine and vice versa. The enzyme also catalyzes the dehydration of D- and L-serine. Both reactions are enhanced by Mg.ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 A resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with L-serine and D-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique "lysino-D-alanyl" residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-D-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.

FIGURE 1.

Covalent modification of the active site. The catalytic Lys-57 in spSRw is converted to lysino-d-alanyl residue. The α-amino group (indicated with “α”) of the d-alanyl moiety in the residue acts as a catalytic base in spSRm. The circled P is a phosphate group.

FIGURE 2.

Dimeric and monomeric structures of spSR. A, stereo view of the spSRw complex with AMP-PCP in the open form along the two-fold axis. The small and large domains of one subunit are shown in light and deep blue, respectively, and the other subunit is shown in green. A large deep groove is formed at the domain interface. The cofactor PLP shown by a space-filling model (yellow) is bound to the bottom of the groove. The groove extends to the subunit interface. AMP-PCP (red) is bound to the groove located at the boundary between the domain interface and the subunit interface on the right or left side of the molecule. N-ter, N terminus; C-ter, C terminus. B, side view of the molecule perpendicular to the two-fold axis. The image structure is turned 90° counterclockwise around the vertical axis relative to the image in Fig. 1A. The groove embracing AMP-PCP is further extended to the subunit interface formed at the back of the molecule. Binding of AMP-PCP induces the rotation of both subunits to widen the back groove. C, stereo view of the subunit in the open form with the secondary structure assignments. The small and the large domains are shown in light and deep blue, respectively. The cofactor PLP-Lys-57 Schiff base located at the bottom of the groove is drawn as the stick model. Besides the cofactor, a metal ion (gray circle) is bound to the large domain to stabilize the active site folding. The metal ion is coordinated by the side-chain carboxylates of Glu-208 and Asp-214. D, large (lower half) domain fitting between spSRw in the open form (red) and spSRm in the closed form (green). The large domain superimposition reveals the rotation of the small domain toward the large domain to close the active site.

FIGURE 3.

Stereo view of the active site in spSR. The front and back of each figure show the solvent side (entrance of the active site) and the protein side (bottom of the active site), respectively. The side chains of the active site residues are depicted as stick models. The cofactor is shown in yellow with phosphate in red. The secondary structures of the small and large domains are drawn in light and deep blue, respectively, and the loops are drawn in gray. Water molecules are represented by red circles. A, close-up view of the active site of spSRw in the open form. A simulated annealing omit map contoured at the 1.0 σ level shows the PLP-Lys-57 internal Schiff base structure. The active site is exposed to the solvent and is filled with many water molecules. The water molecules numbered W1, W2, and W3 are conserved in spSRm in the closed form. B, close-up view of the spSRm complex with the substrate serine. PLP and the lysino-d-alanyl residue form a Schiff base. l-Serine (pink) is tentatively modeled into the active site. Arg-133, which is far from the active site in the open form, approaches the re-face side of PLP to form a salt bridge with the carboxylate of the substrate. The asparagine loop (Ser-81–Ser-82–Gly-83–Asn-84–His-85) moves to the active site center to recognize the carboxylate of the d-alanyl portion of the lysino-d-alanyl residue.

FIGURE 4.

Schematic diagram showing hydrogen bond and salt bridge interactions of the active site residues in spSRm·serine. Putative interactions are shown by dotted lines if the acceptor and donor are less than 3.3 Å apart.

FIGURE 5.

Interactions of AMP-PCP with spSRw in the open form. The simulated annealing omit map is contoured at the 1.0 σ level. AMP-PCP interacts with the small and large domain residues (gray sticks with red oxygen and blue nitrogen) of one subunit and the large domain residues (orange sticks) of the other subunit. Hydrogen bonds are shown by dotted lines (green). Met-53, Asn-84, Gln-87, Glu-281, and Asn-311 (blue sticks) form a hydrogen bond network (red) to link PLP and AMP-PCP. The magnesium ion is surrounded by β- and γ-phosphate and four water molecules. The water molecules are involved in direct hydrogen bonds with the neighboring amino acid residues.

FIGURE 6.

Superposition of external aldimine models between spSRw·d-serine and spSRw·l-serine. The models were designed by taking advantage of the active site superimposition of the rat serine dehydratase complex with O-methyl-l-serine onto spSRm. The substrates, l-serine and d-serine, are shown in green and pink, respectively. The catalytic bases, Lys-57 and Ser-82 (blue), reside on the si- and re-faces of PLP, respectively, and are directed toward the Cα hydrogen atoms. The putative hydrogen bonds between PLP-phosphate and substrate are shown by dotted lines.

FIGURE 7.

The proposed reaction mechanism of spSRw and spSRm with the substrate l- or d-serine.