Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase.

The product of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive anti-infective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of d-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closed-to-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active site in the open conformation of the postdecarboxylation state. In this study, we show that substitution at H49 and H299 negatively impacts LThDP formation by shifting the conformational equilibrium of DXP synthase toward an open conformation. We also present a method for monitoring the dynamics of the spoon motif that uncovered a previously undetected role for H49 in coordinating the closed conformation. Overall, our results suggest that H49 and H299 are critical for the closed, predecarboxylation state providing the first direct link between catalysis and conformational dynamics.

Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures.

The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.