Cofactors of serine racemase that physiologically stimulate the synthesis of the N -methyl- d -aspartate (NMDA) receptor coagonist d -serine

Abstract

High levels of D-serine occur in the brain, challenging the notion that D-amino acids would not be present or play a role in mammals. D-serine levels in the brain are even higher than many L-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others. D-serine is synthesized by a serine racemase (SR) enzyme, which directly converts L-to D-serine. We now report that SR is a bifunctional enzyme, producing both D-serine and pyruvate in cultured cells and in vitro. Transfection of SR into HEK 293 cells elicits synthesis of D-serine and augmented release of pyruvate to culture media. We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate D-serine production by SR and elicit production of pyruvate. Experiments with recombinant enzyme reveal that Mg 2؉ and ATP present in the cell extracts are physiological cofactors and increase 5-to 10-fold the rates of racemization and production of pyruvate. As much as three molecules of pyruvate are synthesized for each molecule of D-serine produced by SR. This finding constitutes a previously undescribed mechanism underlying D-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria. Our data link the production of D-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.glutamate receptors ͉ pyruvate ͉ astrocytes I n recent years, it has been shown that astrocytes and neurons exhibit a dynamic bidirectional signaling that profoundly influences neuronal activity and development (1). Astrocytes modulate synaptic transmission by releasing chemical transmitters and eliciting Ca 2ϩ waves in nearby neurons. The search for new transmitter molecules in the brain uncovered the presence of high levels of D-serine in astrocytes, a D-amino acid not previously thought to occur in mammals (2, 3). D-serine is synthesized by a glial-enriched enzyme serine racemase (SR), which directly converts L-to D-serine (4-7). SR does not bear significant homology with bacterial racemases, and little is known about the mechanism and regulation of D-serine production.D-serine released from astrocytes seems to be an endogenous ligand of the N-methyl-D-aspartate (NMDA) receptor (3,8). Depletion of endogenous D-serine in slices and cultured cells strongly diminishes NMDA receptor responses measured biochemically and electrophysiologically (8). Massive stimulation of NMDA receptors is implicated in neural damage after stroke (9), and inhibitors of SR may be useful to prevent stroke damage. Thus, inhibitors of SR provide a strategy to decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.To clarify the role of D-serine as a modulator of NMDA receptors, one should identify the factors that regulate SR and D-serine signaling. In this paper, we explored mechanisms regulating the production of D-serine by SR. We demonstrate that SR is a unique bifunctional enzyme, produ...

Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149

“…Addition of 1 mM EDTA abolished both dehydration and racemization activities. These results were consistent with a previous study showing that the activities of mammalian SRs are enhanced by Mg 2+ , MgATP and other divalent cations and inhibited completely by EDTA (De Miranda et al, 2002).…”

Serine racemases from barley, Hordeum vulgare L., and other plant species represent a distinct eukaryotic group: Gene cloning and recombinant protein characterization☆

“…The specific activities of racemization and dehydration for 1b were as follows: 14 and 228 nmol/min/mg with regard to HvSR, 10 and 214 nmol/min/mg with regard to OsSR, and 4.1 and 86 nmol/min/mg with regard to AtSR (Fujitani et al, 2006), respectively: the corresponding dehydratase activities were about 20-fold higher than racemase activity. This is as contrast to the dehydration/racemization ratio of about 0.7 for mouse serine racemase (De Miranda et al, 2002). These results thus suggest that the main physiological function of plant serine racemase might be to degrade 1a rather than produce 1a.…”

“…4). The purified HvSR was incubated with 5 µM PLP and 1 mM CaCl 2 which activates AtSR and mammalian serine racemases (Cook et al, 2002;De Miranda et al, 2002;Strisovsky et al, 2003), and the formation of 1a and 2 was determined. The specific activities for racemization of L-serine (1b) to 1a, and dehydration of 1b to 2, were 14 and 228 nmol/min/mg, respectively, and those for racemization of 1a to 1b, and dehydration of 1a to 2, were 2.2 and 89 nmol/min/mg, respectively…”

“…The 1b solution used in the enzyme assay was pre-treated with D-amino acid oxidase and catalase to remove any contaminating 1a according to De Miranda et al (2002). L-Amino acid oxidase was used to remove contaminating 1b in the 1a solution.…”

“…Mammalian serine racemase is an unique enzyme that catalyzes not only serine (1a, 1b) racemization but also dehydration of 1a and 1b to pyruvate (2) in the presence of divalent cations; this distinguishes it from bacterial amino acid racemases (Cook et al, 2002;De Miranda et al, 2002;Strisovsky et al, 2003;Foltyn et al, 2005) (Fig. 1).…”