Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

Abstract

D-Serine is an endogenous coagonist for the N-methyl-D-as-partate 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 Mg⅐ATP 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 Å 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 wildtype enzyme.D-Serine, which is present at a high level in the mammalian brain, serves as an endogenous coagonist for the N-methyl-Daspartate (NMDA) 5 receptor selectively localized on the postsynaptic membrane of the excitatory synapse (1-5) and is involved in excitatory neurotransmission and higher brain functions such as learning and memory (3, 6, 7). Stimulation of the NMDA receptor requires the binding of D-serine as well as the agonist L-glutamate. The major enzyme for D-serine synthesis from L-serine in the brain is considered to be pyridoxal 5Ј-phosphate (PLP)-dependent serine racemase (SR) (8 -10). D-Serine and SR are localized on protoplasmic astrocytes that have the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. Glutamate released from presynaptic neurons approaches and activates the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, which in turn induces SR to produce D-serine and is followed by D-serine release from astrocytes that act on the NMDA receptor. Recently, it was shown that not only glia but also neurons synthesize and release D-serine involved in signaling (11). SR also catalyzes ␣,␤-elimination of water from D-or L-serine to form pyruvate and ammonia as well as the conversion of L-serine into D-serine and vice versa and is presumed to link D-serine synthesis and energy metabolism of astrocytes (12) and to control the D-serine level (13). Mg⅐ATP, which is fully bound to SR under physiologi...

Crystal Structure of a Zinc-dependent d-Serine Dehydratase from Chicken Kidney

“…Some PLP-dependent enzymes also require a metal ion for their enzyme activities. However, unlike chDSD, these enzymes have the activity-relevant metal ion outside of their active site, thus it is proposed that these metal ions are essential to stabilize the catalytically competent conformation of the enzyme (structural cofactors) and are not directly involved in the catalytic reaction (34,35). To investigate whether the zinc ion is required to maintain the structure of catalytically competent chDSD, we determined the crystal structure of the EDTA-treated chDSD ( Table 1).…”

“…The requirement of a metal ion for the enzyme activity is prevalent among PLP-dependent enzymes. Because in all the atomic resolution structures reported hitherto a metal ion is found outside of the active site and this ion has been considered to act as a structural cofactor that stabilizes the catalytically competent conformation of the respective enzymes (34,35). However, the present biochemical and crystallographic analyses strongly suggest that the zinc ion in chDSD is directly involved in the catalytic reaction; a zinc ion is located in the active site and can coordinate the hydroxyl group of the substrate during the catalytic reaction (Fig.…”

“…Structural Basis of Reaction Specificity-chDSD shows no detectable levels of serine racemase activity, whereas serine racemases from mammals and Schizosaccharomyces pombe (spSR) show significant serine dehydratase activity (30,34). To understand the structural basis of the lack of racemase activity of chDSD, we compared the active site structures of chDSD and spSR.…”

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

“…It is known that an optimal hydrophilic-lipophilic balance (HLB) may enable best penetration across Gram-negative outer membrane [33][34][35] ; therefore, a polar moiety lowering the ClogP can be used to facilitate penetration through S. typhimurium cell wall; (b) serine racemase, a PLPdependent enzyme that shows a good structural similarity with OASS, is inhibited by a series of dicarboxylic derivatives such as modified malonates 36,37 ; (c) the synthetic protocol allowing to prepare such compounds is well established and high structural variability can be obtained starting from easily available materials. First, we synthesized two close analogs of 30, which are compounds 25 and 21, in order to assess whether the carboxylic moiety would better work as an ester or an acid.…”

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

“…While the structure of a SR orthologue from the fission yeast Schizosaccharomyces pombe (35.1% 14 or 40% 15 sequence identity to human SR) has been solved 14,16 , the structure of a mammalian SR orthologue remains elusive. We therefore set out to establish a strategy for the generation and screening of human SR mutants in hopes of gaining insight into the structure-function relationships within the enzyme.…”