Validating the inhibitory effects of d- and l-serine on the enzyme activity of d-3-phosphoglycerate dehydrogenases that are purified from Pseudomonas aeruginosa, Escherichia coli and human colon.

BACKGROUND: We previously demonstrated that the serA gene is associated with bacterial pathogenicity, including bacterial penetration through the Caco-2 cell monolayers, bacterial motility, bacterial adherence, and fly mortality. l-Serine is known to inhibit the d-3-phosphoglycerate dehydrogenase (PGDH) activity of the SerA protein, and it significantly reduced the bacterial pathogenicity as described above. We also demonstrated that in a PGDH assay using crude extracts isolated from overnight cultures of E. coli overexpressing the P. aeruginosa serA gene, l-serine inhibited the PGDH activity of the SerA protein. The basal PGDH activity of the negative control strain was high, presumably due to contamination of unknown proteins in the crude extracts. Therefore, to further confirm the direct inhibition of PGDH activity of P. aeruginosa SerA by l-serine, we purified and characterized the PGDH from P. aeruginosa and compared it with the previously characterized PGDHs from E. coli, and the human colon as controls. RESULTS: Optimum pH and ionic strength of the purified PGDHs were different depending on the three species; optimal activity of P. aeruginosa PGDH was at pH 7.5 with 50-100 mM Tris-HCl, E. coli PGDH was at pH 8.5 with 100-200 mM Tris-HCl, and human PGDH was at pH 9.0 with 100-200 mM Tris-HCl. The addition of l-serine reduced the activity of PGDH from P. aeruginosa and E. coli, but not the PGDH from human colon. The median inhibitory concentration (IC50) of l-serine was 630 µM for P. aeruginosa and 250 µM for E. coli, while IC50 of d-serine was much higher than that of l-serine; 76 mM in P. aeruginosa PGDH and 45 mM in E. coli PGDH. CONCLUSIONS: These results suggest that l-serine significantly repressed P. aeruginosa pathogenicity through direct inhibition of the PGDH activity, but was not able to inhibit the human PGDH activity. Oral administration of l-serine to compromised hosts might interfere with bacterial translocation and prevent gut-derived sepsis caused by P. aeruginosa through inhibition of the function of the serA gene product.

Enantioselective analysis of D- and l- Serine on a layer-by-layer imprinted electrochemical sensor.

The present work describes a new, simple, and easy method of generating acrylamide functionalised reduced graphene oxide-fullerene layer-by-layer assembled dual imprinted polymers to quantify D- and L-Serine at ultra trace level in aqueous and real samples. Herein, the pencil graphite electrode was initially spin coated with D-Serine imprinted acrylamide functionalized reduced graphene oxide. After 10 min thermal treatment (50 °C), this electrode was again modified with L-Serine imprinted acrylamide functionalized fullerene molecules. This bilayer assembly was finally made thermally stable by 60 °C exposure for 3 h. The proposed sensor showed better electronic properties with an improved synergism. We have compared this modified electrode with other modified pencil graphite electrodes like single layered acrylamide functionalised reduced graphene oxide or fullerene, single layered acrylamide functionalised reduced graphene oxide-fullerene composite and double layered acrylamide functionalised reduced graphene oxide or fullerene molecules, which yielded very inferior sensitivity due to possible agglomeration and decreased synergism. The chosen system demonstrated a very good analytical figures of merit with differential pulse anodic stripping voltammetry and cyclic voltammetry transduction, showing lower limits of detection (0.24 ng mL-1, S/N = 3) for both isomers. The proposed sensor assures practical applications as disease biomarker, manifesting several diseases at very ultra-trace level.

Bioanalytical method for the simultaneous determination of D- and L-serine in human plasma by LC/MS/MS.

D-Serine is an endogenous modulator of N-methyl-D-aspartate (NMDA) receptors. Plasma concentrations of D-serine and the ratio of D-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the D- and L-isomers of serine in human plasma. Since D- and L-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. D- and L-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. D-Serine (m/z 106.1 > 60.0), L-serine (m/z 106.1 > 60.1) and DL-serine-d3 (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of D- and L-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01-10 µg/mL (D-serine) and 0.1-100 µg/mL (L-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of D- and L-serine in human plasma were 0.124-0.199 and 7.97-13.1 µg/mL, respectively.