Mode of action of lipoprotein modification enzymes-Novel antibacterial targets.

Lipoproteins are characterized by a fatty acid moiety at their amino-terminus through which they are anchored into membranes. They fulfill a variety of essential functions in bacterial cells, such as cell wall maintenance, virulence, efflux of toxic elements including antibiotics, and uptake of nutrients. The posttranslational modification process of lipoproteins involves the sequential action of integral membrane enzymes and phospholipids as acyl donors. In recent years, the structures of the lipoprotein modification enzymes have been solved by X-ray crystallography leading to a greater insight into their function and the molecular mechanism of the reactions. The catalytic domains of the enzymes are exposed to the periplasm or external milieu and are readily accessible to small molecules. Since the lipoprotein modification pathway is essential in proteobacteria, it is a potential target for the development of novel antibiotics. In this review, we discuss recent literature on the structural characterization of the enzymes, and the in vitro activity assays compatible with high-throughput screening for inhibitors, with perspectives on the development of new antimicrobial agents.

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most γ-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein Apolipoprotein N-acyltransferase (Lnt).

Lipoprotein modification is an essential process in Gram-negative bacteria. The action of three integral membrane proteins that catalyze the transfer of fatty acids derived from membrane phospholipids or cleave the signal peptide of the lipoprotein substrate result in the formation of mature triacylated proteins. Inactivation of the enzymes leads to mis-localization of immature lipoproteins and consequently cell death. Biochemical studies and the development of in vitro assays are challenging due to the fact that the enzymes and substrates are all membrane-embedded proteins difficult to overproduce and purify. Here we describe a sensitive fluorescence-based assay to monitor bacterial apolipoprotein N-acyltransferase activity.

Identification of Lipoproteins Using Globomycin and Radioactive Palmitate.

Bacterial lipoproteins are characterized by fatty acids that are covalently attached to their amino terminus via posttranslational modification in the cytoplasmic membrane. Three enzymatic steps are involved in the synthesis of mature triacylated lipoprotein: prolipoprotein converts into diacylglyceryl-prolipoprotein that in turn converts into apolipoprotein, which is finally converted into mature triacylated lipoprotein. Here we describe the detection of one of these intermediate forms of lipoprotein, diacylglyceryl-prolipoprotein, using 3H-palmitate labeling and inhibition by globomycin and detection by fluorography.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein's invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

The molecular mechanism of bacterial lipoprotein modification--how, when and why?

Posttranslational modification of proteins by lipidation is a common process in biological systems. Lipids provide protein stability, interaction with other membrane components, and in some cases, due to reversibility of the process, a mechanism for regulating protein localization and function. Bacterial lipoproteins possess fatty acids at their amino-termini that are derived from phospholipids, and this lipid moiety anchors the proteins into the membrane. These lipids, as is the case for lipopolysaccharides and lipoteichoic acids, play an important role in signaling of the innate immune system through the interaction with Toll-like receptors. Over the past three years, tremendous progress has been made in understanding the mechanism by which lipoproteins become lipidated. Advanced methodology in mass spectrometry, proteomics and genome-wide analyses allowed precise characterization of lipoprotein modifications and the identification of the enzymes catalyzing the reactions in diverse bacterial species. This review will highlight new findings on bacterial lipoprotein modification with focus on the reaction mechanisms and the role of lipoproteins in cell envelope homeostasis.

Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction.

Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation, led to the formation of the thioester acyl-enzyme intermediate and to the accumulation of apo-Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cytoplasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N-acylation of lipoproteins.

Phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.

Lgt of Escherichia coli catalyzes the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth, as demonstrated here by the analysis of an lgt depletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein. Its membrane topology was determined by fusing Lgt to ß-galactosidase and alkaline phosphatase and by substituted cysteine accessibility method (SCAM) studies. The data show that Lgt is embedded in the membrane by seven transmembrane segments, that its N terminus faces the periplasm, and that its C terminus faces the cytoplasm. Highly conserved amino acids in Lgt of both Gram-negative and Gram-positive bacteria were identified. Lgt enzymes are characterized by a so-called Lgt signature motif in which four residues are invariant. Ten conserved residues were replaced with alanine, and the activity of these Lgt variants was analyzed by their ability to complement the lgt depletion strain. Residues Y26, N146, and G154 are absolutely required for Lgt function, and R143, E151, R239, and E243 are important. The results demonstrate that the majority of the essential residues of Lgt are located in the membrane and that the Lgt signature motif faces the periplasm.

Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.

The enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein. To gain insight into the catalytic mechanism of this two-step reaction, we overproduced and purified the enzyme of Escherichia coli and studied its N-acyltransferase activity using a novel in vitro assay. The purified enzyme was fully active, as judged by its ability to form a stable thioester acyl-enzyme intermediate and N-acylate the apo-form of the murein lipoprotein Lpp in vitro. Incorporation of [(3)H]palmitate and mass spectrometry analysis demonstrated that Lnt recognized the synthetic diacylglyceryl-modified lipopeptide FSL-1 as a substrate in a mixed micelle assay. Kinetics of Lnt using phosphatidylethanolamine as an acyl donor and FSL-1 as a substrate were consistent with a ping-pong type mechanism, demonstrating slow acyl-enzyme intermediate formation and rapid N-acyl transfer to the apolipopeptide in vitro. In contrast to earlier in vitro observations, the N-acyltransferase activity was strongly affected by the phospholipid headgroup and acyl chain composition.

The essential Escherichia coli apolipoprotein N-acyltransferase (Lnt) exists as an extracytoplasmic thioester acyl-enzyme intermediate.

Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerophospholipid to the free alpha-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl-enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of the active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage. Lnt(C387A) did not form an acyl-enzyme intermediate and, like Lnt(C387S), did not have any detectable Lnt activity, indicating that acylation cannot occur at other positions in the catalytic domain. The existence of this thioacyl-enzyme intermediate allowed us to determine whether essential residues in the catalytic domain of Lnt affect the first step of the reaction, the formation of the acyl-enzyme intermediate, or the second step in which the acyl chain is transferred to the apolipoprotein substrate. In the catalytic triad, E267 is required for the formation of the acyl-enzyme intermediate, indicating its role in enhancing the nucleophilicity of C387. E343 is also involved in the first step but is not in close proximity to the active site. W237, Y388, and E389 play a role in the second step of the reaction since acyl-Lnt is formed but N-acylation does not occur. The data presented allow discrimination between the functions of essential Lnt residues in catalytic activity and substrate recognition.

Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane.

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

Identification of essential residues in apolipoprotein N-acyl transferase, a member of the CN hydrolase family.

Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of E. coli are conserved in these proteins.

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM.

The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

Traffic spotting: poles apart.

Finding out where specific functions are carried out within a bacterial cell has now become technically feasible. Here we consider recent experiments aimed at determining where bacteria translocate proteins across the cytoplasmic membrane using the Sec machinery.

A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region.

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region.

Assembly of cell division proteins at the E. coli cell center.

Cell division in Escherichia coli requires the coordinated action of at least ten proteins. In recent years, substantial progress has been made in understanding the assembly of these proteins at the cell septum. These findings suggest a largely stepwise appearance of cell division proteins at the centre of the cell.