With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.

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.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.Bacteria and humans have co-evolved for millennia, and many examples exist of how various symbiotic and commensal partnerships contribute to human health and nutrition ranging from the metabolism of complex carbohydrates to the provision of vital micronutrients (1). Queuosine is an example of a micronutrient, synthesized exclusively by bacteria but which, for poorly defined reasons, is utilized by almost all eukaryotic species with the exception of the baker's yeast, Saccharomyces cerevisiae (2).Bacterial queuosine biosynthesis occurs in two stages. First, a series of five enzymatic steps convert guanosine triphosphate nucleoside (GTP) to the soluble 7-aminomethyl-7-deazaguanine molecule. Subsequently, 7-aminomethyl-7-deazaguanine is inserted into the wobble position of tRNA containing a GUN consensus sequence (Tyr, Asp, Asn, and His) by means of the single enzyme species, tRNA guanine transglycosylase (TGT), and is further remodeled in situ to queuosine (3). Eukaryotes must acquire queuosine or its free nucleobase, queuine, from food and the gut microflora. Curiously, both cytosolic and mitochondrial tRNA species are modified by queuosine (2). The eukaryotic enzyme that performs this reaction, queuine tRNA ribosyltransferase, has recently been identified as a heterodimeric complex, consisting of the eukaryotic homologue of the catalytic TGT subunit and a related protein called queuine tRNA ribosyltransferase domain containing 1 (QTRTD1), both of which localize to the mitochondria (4, 5).Stu...

tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA Asp , tRNA Asn , tRNA His , and tRNA Tyr ; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle. Queuosine (Q 3 ; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNA Tyr , tRNA Asn , tRNA His , and tRNA Asp (1). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2, 3). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4) and opossum (5). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNA Asp and a galactosyl group to Q-tRNA Tyr (1). Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ 1 ) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5Ј-phosphate subsequent to normal tRNA turnover (7). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9, 10).The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (␤/␣) 8 TIM barrel with a C-terminal zinc-binding subdomain (11). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme...

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases [1][2][3][4][5] , has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field 6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting 22,23 . Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2) 24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies 4,26 . The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases. Video LinkThe video component of this article can be found at https://www.jove.com/video/4001/ Protocol 1. Laboratory Set-up Pre-harvesting 1. In preparation for harvesting, fill the dry foam Dewar with liquid nitrogen and place it beside the microscope where harvesting is to take place. 2. Submerge the storage puck, open end up, in the liquid nitrogen inside the foam Dewar and allow it to fully cool. 3. Secure a micro-mount of a size...