The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis.

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that LpxE structurally resembles YodM in , a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak activity on C-PP, and rescues deficient in PGP and C-PP phosphatase activities; deletion of in reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C-PP phosphatase activity. Suppression of plasmid-borne in deficient in chromosomally encoded C-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death.

Structure-Activity Relationship of Sulfonyl Piperazine LpxH Inhibitors Analyzed by an LpxE-Coupled Malachite Green Assay.

The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast majority of Gram-negative pathogens and an excellent novel antibiotic target. The P-radioautographic thin-layer chromatography assay has been widely used for analysis of LpxH activity, but it is inconvenient for evaluation of a large number of LpxH inhibitors over an extended time period. Here, we report a coupled, nonradioactive LpxH assay that utilizes the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for quantitative removal of the 1-phosphate from lipid X, the product of the LpxH catalysis; the released inorganic phosphate is subsequently quantified by the colorimetric malachite green assay, allowing the monitoring of the LpxH catalysis.

Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes.

The other 90% of the protein: assessment beyond the Calphas for CASP8 template-based and high-accuracy models.

For template-based modeling in the CASP8 Critical Assessment of Techniques for Protein Structure Prediction, this work develops and applies six new full-model metrics. They are designed to complement and add value to the traditional template-based assessment by the global distance test (GDT) and related scores (based on multiple superpositions of Calpha atoms between target structure and predictions labeled "Model 1"). The new metrics evaluate each predictor group on each target, using all atoms of their best model with above-average GDT.