Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.

The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Computational Identification of Kinases That Control Axon Growth in Mouse.

The determination of signaling pathways and transcriptional networks that control various biological processes is a major challenge from both basic science and translational medicine perspectives. Because such analysis can point to critical disease driver nodes to target for therapeutic purposes, we combined data from phenotypic screening experiments and gene expression studies of mouse neurons to determine information flow through a molecular interaction network using a network propagation approach. We hypothesized that differences in information flow between control and injured conditions prioritize relevant driver nodes that cause this state change. Identifying paths likely taken from potential source nodes to a set of transcription factors (TFs), called sinks, we found that kinases are enriched among source genes sending significantly different amounts of information to TFs in an axonal injury model. Additionally, TFs found to be differentially active during axon growth were enriched in the set of sink genes that received significantly altered amounts of information from source genes. Notably, such enrichment levels hold even when restricting the set of source genes to only those kinases observed to support or hamper neurite growth. That way, we found a set of 71 source genes that send significantly different levels of information to axon growth-relevant TFs. We analyzed their information flow changes in response to axonal injury and their influences on TFs predicted to facilitate or antagonize axon growth. Finally, we drew a network diagram of the interactions and changes in information flow between these source genes and their axon growth-relevant sink TFs.

High-Throughput Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms.

The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer.

Protein kinase signaling networks stringently regulate cellular processes, such as proliferation, motility, and cell survival. These networks are also central to the evolution and progression of cancer. Accordingly, genetically encoded fluorescent biosensors capable of directly illuminating the spatiotemporal dynamics of kinase signaling in live cells are being increasingly used to investigate kinase signaling in cancer cells and tumor tissue sections. These biosensors enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Herein, we first review common design strategies for kinase activity biosensors, including signaling targets, biosensor components, and fluorescent proteins involved. Subsequently, we discuss applications of biosensors to study the biology and management of cancer. These versatile molecular tools have been deployed to study oncogenic kinase signaling in living cells and image kinase activities in tumors or to decipher the mechanisms of anticancer drugs. We anticipate that the diversity and precision of genetically encoded biosensors will expand their use to further unravel the dysregulation of kinase signaling in cancer and the modes of actions of cancer-targeting drugs.

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase.

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi.

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-ß-d-glucopyranose) in the presence of Mg2+ -ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting ß-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20-50 mm), the apparent binding of levoglucosan (Km =180 mm) and ATP (Km =1.0 mm), as well as the inhibition by ADP (Ki =0.45 mm) and d-glucose-6-phosphate (IC50 =56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4 C1 →1 C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus kodakarensis.

Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. kodakarensis.

Biosynthesis of an antiviral compound using a stabilized phosphopentomutase by multipoint covalent immobilization.

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.

Application of Monoclonal Antibodies to Detect and Compare the Levels of Streptococcus mutans in Adolescents Undergoing Orthodontic Treatment with Those Not Undergoing Treatment.

The purpose of this study was to detect Streptococcus mutans by using monoclonal antibodies (mAbs) against S. mutans that cause dental caries and compare the levels of the bacterium between the saliva of adolescents undergoing orthodontic treatment (OT) and those not undergoing treatment (NT). Saliva samples, collected from 25 OT adolescents (with a mean age of 12.84 years) and 25 NT adolescents (mean age of 12.4 years), were analyzed by Dentocult-SM and enzyme-linked immunosorbent assay using mAbs against Ag I/II (ckAg I/II) and GTF B (ckGTF B), GTF C (ckGTF C), and GTF D (ckGTF D) of S. mutans. The DMFT index was slightly higher in the OT group (5.12 in OT and 4.96 in NT) and the level of S. mutans (≥105 CFU/mL) was higher in OT (72%) than in NT (56%). The detected levels of ckAg I/II, ckGTF B, ckGTF C, and ckGTF D were slightly higher in OT than in NT. The results of this study indicate that use of mAbs against S. mutans yields sensitive detection for the bacterium in saliva samples and shows that it has a reliable connection to the number of S. mutans and decayed, missing, filled teeth (DMFT), suggesting that the levels of S. mutans in saliva can be defined and compared by the application of the mAbs.

Dematin, a human erythrocyte cytoskeletal protein, is a substrate for a recombinant FIKK kinase from Plasmodium falciparum.

P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction.

An integrative approach for the large-scale identification of human genome kinases regulating cancer metastasis.

Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. FROM THE CLINICAL EDITOR: This team of authors have utilized a self-assembled cell microarray to screen genes that regulate cancer cell migration using a human genome siRNA library of kinases. They validated previously known genes and identified novel ones that may serve as therapeutic targets.

Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling.

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

Expression, purification, and characterization of cytokinin signaling intermediates: Arabidopsis histidine phosphotransfer protein 1 (AHP1) and AHP2.

KEY MESSAGE: We have expressed, purified, and biophysically characterized recombinant AHP1 and AHP2. Also, using computational homology models for AHP1, ARR7, and AHP1­ARR7 complex, we identified threedimensional positioning of key amino acids. Cytokinin signaling involves activation of Arabidopsis Response Regulators (ARRs) by Arabidopsis Histidine Phosphotransfer Proteins (AHPs) by phosphorylation. Type-A ARRs are key regulators of several developmental pathways, but the mechanism underlying this phosphorylation and activation is not known in plants. In this study, we report the successful expression and purification of recombinant AHP1 and AHP2. Biophysical characterization shows that these two recombinant proteins were purified to homogeneity and possess well-defined secondary structures. Brief attempts to purify recombinant ARR7 posed problems during size-exclusion chromatography. Nevertheless, we generated computational homology models for AHP1, ARR7, and AHP1-ARR7 complex using crystal structures of homologous proteins from other organisms. The homology models helped to identify the three-dimensional positioning of the key conserved residues of AHP1 and ARR7 involved in phosphorylation. The similarity in positioning of these residues to other homologous proteins suggests that AHPs and type-A ARRs could be structurally conserved across kingdoms. Thus, our homology models can serve as valuable tools to gain structural insights into the phosphorylation and activation of cytokinin response regulators in plants.

Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana.

Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale.

The N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the k(ex) of ~500 s(-1) which compares to the phosphoryl transfer rate of ~850 s(-1) between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins.

Conformational selection and substrate binding regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli enzyme I.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the ß3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ∼1500 s(-1)) between major open (∼97%) and minor closed (∼3%) conformations. Upon PEP binding, the ß3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.

The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate.

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.