Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product-or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPSand UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.cell wall ͉ geranylgeranyl diphosphate synthase ͉ undecaprenyl diphosphate synthase ͉ x-ray structure I soprenoid biosynthesis involves the condensation of C 5 -diphosphates to form a very broad range of compounds used in cell membrane (cholesterol, ergosterol), cell wall (lipid I, II, peptidoglycan) and terpene biosynthesis, electron transfer (quinone, heme a, carotenoid, chlorophyll), and in many eukaryotes, cell signaling pathways (Ras, Rho, Rap, Rac). There has, therefore, been considerable interest in developing specific inhibitors of some of these pathways to modify cell function. For example, the bisphosphonate drugs used to treat bone resorption diseases such as osteoporosis (1) have been thought to function by targeting farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) in osteoclasts, leading to dysregulation of cell-signaling pathways involving small GTPases, and in some parasitic protozoa, leading to inhibition of ergosterol biosynthesis (2). However, in recent work Goffinet et al. (3) proposed that the main biological activity of the most potent bisphosphonate zoledronate (Zometa) in humans cells is directed against protein geranylgeranylation. This opens up the intriguing possibility that it might be possible to enhance potency by developing drugs that work by inhibiting geranylgeranyl diphosphate synthase (GGPPS, EC 2.5.1.30), the enzyme that produces the geranylgeranyl diphosphate (GGPP) used to geranylgeranylate e.g., Rac, Rap, and Rho. Based on the recent observation of a previously uncharacterized (GGPP) inhibitor site in GGPPS (4), we reasoned that larger, more hydrophobic species than those in current use might bind to this site and exhibit enhanced activity, because of increased hydrophobic stabilization and, in cells, enhanced lipophilicity. Here, we thus report structures of a series of five bisphosphonates bound to GGPPS together with, for comparative purposes, the structures of five isoprenoid diphosphate-GGPPS complexes. We find three quite different binding modes, corresponding to FPP/GPP (substrate), IPP (substrate), and GGPP [product/ inhibitor (4)...

The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C pro ) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL pro ) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL pro . As shown in the present study, some of these compounds were also found to be active against 3C pro of CV strain B3 (CVB3). Several crystal structures of 3C pro from CVB3 and 3CL pro from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His 40 -Cys 147 catalytic dyad of CVB3 3C pro . The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Pyridoxine 5Ј-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5Ј-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of ∼0.2 sec −1 and K m values in the low micromolar range for both pyridoxine 5Јphosphate and pyridoxamine 5Ј-phosphate. Pyridoxal 5Ј-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5Ј-phosphate tightly on each subunit.

Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-Å resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 Å 2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His 62 , Cys 91 , and Cys 94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.Cytosine deaminase (CD, 1 EC 3.5.4.1) catalyzes the deamination of cytosine to uracil and 5-methylcytosine to thymine. The enzyme has been found in bacteria and fungi, where it plays an important role in pyrimidine salvage. However, it is not present in mammalian cells, which utilize cytidine deaminase (CDA) instead (1). The bacterial and fungal CDs are distinct from each other and have evolved separately. The 426-residue hexameric Escherichia coli enzyme like the murine adenosine deaminase belongs to the (␤/␣) 8 -barrel amidohydrolase superfamily, in which four histidines and one aspartate located at similar spatial positions are conserved for metal coordination and enzyme catalysis (2-4). On the other hand, the 158-residue dimeric yeast counterpart may share two conserved signature sequences, HXE and CXXC, with a variety of deaminases, and thus has been grouped into the cytidine and deoxycytidylate deaminase family in the Pfam protein family data base (5, 6). The crystal structure of E. coli CDA reveals that the signature sequences contain a zinc binding motif, with histidine and two cysteines acting as zinc ligands while the glutamate serves as a proton shuttle (7).The antimetabolite 5-fluorouracil (5-FU) is one of the most active chemotherapeutic agents for the treatment of colorectal cancer, but it has limited efficacy due to gastrointestinal and hematological toxicities (8). Because of its ability to convert the relatively nontoxic 5-fluorocytosine (5-FC) into 5-FU and its absence in mammalian cells, CD has become an attractive candidate for the reduction of 5-FU toxicity toward n...

"Head-to-head" terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg 2þ cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.triterpene | X-ray crystallography | drug discovery | staphyloxanthin | quinuclidine H ead-to-head terpene synthases catalyze the first committed steps in the biosynthesis of sterols and carotenoid pigments: the C1′-2,3 condensation of two isoprenoid diphosphates to form a cyclopropylcarbinyl diphosphate (1, 2), followed by ring opening to form squalene, dehydrosqualene, or phytoene. In humans and in many pathogenic yeasts, fungi, and protozoa, as well as in plants, the isoprenoid diphosphate is farnesyl diphosphate (FPP) and the initial product is the C 30 isoprenoid, presqualene diphosphate (PSPP). As implied by its name, PSPP is then converted (by the same enzyme as used in the condensation reaction, squalene synthase, SQS) to squalene which, after epoxidation, is cyclized to lanosterol (3), as shown in Fig. 1. Lanosterol then undergoes numerous additional reactions, resulting in formation of sterols, key cell membrane components. As such, squalene synthase inhibitors are of interest as antiparasitics, in particular against Trypanosoma cruzi (4) and Leishmania spp. (5), the causative agents of Chagas disease and the leishmaniases. In plants, the related enzyme phytoene synthase (PSY) catalyzes the condensation of two C 20 isoprenoid diphosphate (geranylgeranyl diphosphate, GGPP) molecules (6) to form prephytoene diphosphate (PPPP) that, after ring opening, forms phytoene, which is then converted to carotenoid pigments (7) (Fig. 1). In the bacterium Staphylococcus aureus, the initial step in formation of the carote...

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.cryoelectron microscopy | mass spectrometry | protein glycosylation | alphacoronavirus | feline infectious peritonitis virus C oronaviruses (CoVs) are enveloped viruses with single-