The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4 substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4 of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.Pyridoxal 5Ј-phosphate (PLP) serves as a cofactor for many enzymes involved in amino acid and sugar metabolism. In many bacteria and plants, PLP is synthesized by a de novo pathway, but most cells rely on a nutritional source of vitamin B 6 , i.e., pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM) (24). Most cell types have a salvage pathway for reutilizing the PL liberated during protein turnover (27). The salvage pathway involves an ATP-dependent pyridoxal kinase that phosphorylates PL, PN, and PM by transferring the terminal phosphate of ATP to the 5Ј-hydroxyl group of these substrates. The product of PN and PM phosphorylation is converted to PLP by pyridoxine 5Ј-phosphate oxidase, which is also expressed in most cells. The PL kinases that have been purified and studied show activity with PN and PM, in addition to PL, and are often referred to as PL/PN/PM kinases (14, 15). For brevity, we will refer to these enzymes as PL kinases with the understanding that many of them exhibit considerable activity with PN and PM.PL kinase has been purified from bacterial, plant, and mammalian sources, and evidence suggests that most organisms contain a single PL kinase, coded by a pdxK gene. However, studies with Escherichia coli mutants that were blocked in the de novo biosynthetic pathway and with an inactivated pdxK gene, which codes for E. coli pyridoxal kinase 1 (ePL kinase 1), were still able to grow on pyridoxal (26). A search for a gene that expressed a protein that had PL kinase activity in E. coli led to the discovery of the pdxY gene (26). The E. coli protein ePL kinase 2, expressed by this gene, was shown to have some PL kinase act...

Molecular replacement (MR) is the method of choice for X-ray crystallography structure determination when structural homologues are available in the Protein Data Bank (PDB). Although the success rate of MR decreases sharply when the sequence similarity between template and target proteins drops below 35% identical residues, it has been found that screening for MR solutions with a large number of different homology models may still produce a suitable solution where the original template failed. Here we present the web tool CaspR, implementing such a strategy in an automated manner. On input of experimental diffraction data, of the corresponding target sequence and of one or several potential templates, CaspR executes an optimized molecular replacement procedure using a combination of well-established stand-alone software tools. The protocol of model building and screening begins with the generation of multiple structure-sequence alignments produced with T-COFFEE, followed by homology model building using MODELLER, molecular replacement with AMoRe and model refinement based on CNS. As a result, CaspR provides a progress report in the form of hierarchically organized summary sheets that describe the different stages of the computation with an increasing level of detail. For the 10 highest-scoring potential solutions, pre-refined structures are made available for download in PDB format. Results already obtained with CaspR and reported on the web server suggest that such a strategy significantly increases the fraction of protein structures which may be solved by MR. Moreover, even in situations where standard MR yields a solution, pre-refined homology models produced by CaspR significantly reduce the time-consuming refinement process. We expect this automated procedure to have a significant impact on the throughput of large-scale structural genomics projects. CaspR is freely available at http://igs-server.cnrs-mrs.fr/Caspr/.

Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy. adaptive evolution | NADPH metabolism | complex I E volutionary microbiology under environmental or metabolic constraints is an emergent field of research. Although it is known that growing microorganisms are not static over either short or long periods, the evolutionary process called adaptive evolution developed to adapt to constraints is poorly understood at the genetic, biochemical, and metabolic levels.Experiments of adaptive evolution have been conducted in the laboratory to understand the behavior of Escherichia coli in response to different environmental or metabolic constraints: (i) wide serial subculturing for over 20 years (more than 40,000 generations) on glucose-limited mineral medium (1), (ii) growth for more than 700 generations on glycerol, a maladapted substrate although a complete metabolic pathway for this substrate was already present (2), and (iii) culture over 600-800 generations of single gene deletion mutants affected in metabolic key branch points [phosphoglucose isomerase (Δpgi), phosphoenolpyruvate carboxylase (Δppc), triose phosphoisomerase (Δtpi), phosphotransacetylase (Δpta)] to evaluate the effect of targeted metabolic perturbations on the structure of the metabolic network (3). The main conclusions of this work highlighted the strong robustness of the genetic and metabolic networks of E. coli and indicated that evolution never invented a new solution such as a reassignment of enzyme function to adapt to the environmental or metabolic constraints. Instead, the main feature of evolution was an increased in the capacity of already active pathways or the activation of latent pathways.The objective of the present work was to study the ability of E. coli to adapt to and overcome a strong unbalance and an imposed rigidity of the redox state of cellular metabo...

Caspase‐4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non‐canonical inflammasome, which drives inflammatory responses during Gram‐negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome‐wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS‐mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase‐4 levels in human monocytes and iPSC‐derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non‐canonical inflammasome. Upon IFN‐γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase‐4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram‐negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non‐canonical inflammasome.