Ethyl 2-(3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl-oxy)acetate.

The title compound, C(12)H(14)O(6), features a roughly planar mol-ecule (r.m.s. deviation for all non-H atoms = 0.287 Å). In the crystal, the mol-ecules are held together by C-H⋯O hydrogen bonds.

Fabry disease: identification of 50 novel alpha-galactosidase A mutations causing the classic phenotype and three-dimensional structural analysis of 29 missense mutations.

Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase, alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The molecular lesions in the alpha-Gal A gene causing the classic phenotype of Fabry disease in 66 unrelated families were determined. In 49 families, 50 new mutations were identified, including: 29 missense mutations (N34K, T41I, D93V, R112S, L166G, G171D, M187T, S201Y, S201F, D234E, W236R, D264Y, M267R, V269M, G271S, G271V, S276G, Q283P, A285P, A285D, M290I, P293T, Q312H, Q321R, G328V, E338K, A348P, E358A, Q386P); nine nonsense mutations (C56X, E79X, K127X, Y151X, Y173X, L177X, W262X, Q306X, E338X); five splicing defects (IVS4-1G>A, IVS5-2A>G, IVS5+3A>G, IVS5+4A>G, IVS6-1G>C); four small deletions (18delA, 457delGAC, 567delG, 1096delACCAT); one small insertion (996insC); one 3.1 kilobase Alu-Alu deletion (which included exon 2); and one complex mutation (K374R, 1124delGAG). In 18 families, 17 previously reported mutations were identified, with R112C occurring in two families. In two classically affected families, affected males were identified with two mutations: one with two novel mutations, D264Y and V269M and the other with one novel (Q312H) and one previously reported (A143T) mutation. Transient expression of the individual mutations revealed that D264Y and Q312H were localised in the endoplasmic reticulum and had no detectable or markedly reduced activity, whereas V269M and A143T were localised in lysosomes and had approximately 10 per cent and approximately 35 per cent of expressed wild-type activity, respectively. Structural analyses based on the enzyme's three-dimensional structure predicted the effect of the 29 novel missense mutations on the mutant glycoprotein's structure. Of note, three novel mutations (approximately 10 per cent) were predicted not to significantly alter the glycoprotein's structure; however, they were disease causing. These studies further define the molecular heterogeneity of the alpha-Gal A mutations in classical Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and provide insights into the structural alterations of the mutant enzymes that cause the classic phenotype.

A snapshot of viral evolution from genome analysis of the tectiviridae family.

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.

The receptor binding protein P2 of PRD1, a virus targeting antibiotic-resistant bacteria, has a novel fold suggesting multiple functions.

Bacteriophage PRD1 is unusual, with an internal lipid membrane, but has striking resemblances to adenovirus that include receptor binding spikes. The PRD1 vertex complex contains P2, a 590 residue monomer that binds to receptors on antibiotic-resistant strains of E. coli and so is the functional counterpart to adenovirus fiber. P2 structures from two crystal forms, at 2.2 and 2.4 A resolution, reveal an elongated club-shaped molecule with a novel beta propeller "head" showing pseudo-6-fold symmetry. An extended loop with another novel fold forms a long "tail" containing a protruding proline-rich "fin." The head and fin structures are well suited to recognition and attachment, and the tail is likely to trigger the processes of vertex disassembly, membrane tube formation, and subsequent DNA injection.

Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon.

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 A resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 (a=b=157.8 A, c=114.2 A, gamma=120 degrees) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large approximately 100 A diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.

Heteroarotinoids with anti-cancer activity against ovarian cancer cells.

The Flex-Het compound 10a (SHetA2-NSC 721689) {[4-nitrophenylamino][(2,2,4,4-tetramethylthiochroman-6-yl)amino]methane-1-thione]} has shown promise in preclinical testing as an anti-cancer agent without evidence of toxicity, skin irritancy, or teratogenicity. One objective of this study was to synthesize a series of heteroarotinoids structurally related to SHetA2 and to measure the effect of structural alterations on the cytotoxicity activities of the compounds on A2780 ovarian cancer cells. Alterations included comparisons of activity of an NO2 end group versus a CO2Et end group, a thiourea linker versus a urea linker, and a distorted, thiochroman ring unit versus a planar quinoline ring unit. Cytotoxicity assays demonstrated the thiourea linker compounds to be similar in potency to the urea linker counterparts, the NO2 substitutions were slightly more potent than the CO2Et substitutions, and replacement of the thiochroman group with a planar quinoline fused ring system markedly reduced activity. The mechanism of cytotoxicity through apoptosis was confirmed for the compounds. The optimal combination of structural features for enhancing potency consisted of a urea linker, a NO2 substitution, and a flexible thiochroman unit. Extensive H-bonding in the more active urea derivative was confirmed by X-ray and NMR analyses. This is the first example in which the biological activity of flexible, thiochroman units is compared to that of fused aryl units in a heteroarotinoid molecule.

The X-ray crystal structure of P3, the major coat protein of the lipid-containing bacteriophage PRD1, at 1.65 A resolution.

P3 has been imaged with X-ray crystallography to reveal a trimeric molecule with strikingly similar characteristics to hexon, the major coat protein of adenovirus. The structure of native P3 has now been extended to 1.65 A resolution (R(work) = 19.0% and R(free) = 20.8%). The new high-resolution model shows that P3 forms crystals through hydrophobic patches solvated by 2-methyl-2,4-pentanediol molecules. It reveals details of how the molecule's high stability may be achieved through ordered solvent in addition to intra- and intersubunit interactions. Of particular importance is a 'puddle' at the top of the molecule containing a four-layer deep hydration shell that cross-links a complex structural feature formed by 'trimerization loops'. These loops also link subunits by extending over a neighbor to reach the third subunit in the trimer. As each subunit has two eight-stranded viral jelly rolls, the trimer has a pseudo-hexagonal shape to allow close packing in its 240 hexavalent capsid positions. Flexible regions in P3 facilitate these interactions within the capsid and with the underlying membrane. A selenometh-ionine P3 derivative, with which the structure was solved, has been refined to 2.2 A resolution (R(work) = 20.1% and R(free) = 22.8%). The derivatized molecule is essentially unchanged, although synchrotron radiation has the curious effect of causing it to rotate about its threefold axis. P3 is a second example of a trimeric 'double-barrel' protein that forms a stable building block with optimal shape for constructing a large icosahedral viral capsid. A major difference is that hexon has long variable loops that distinguish different adenovirus species. The short loops in P3 and the severe constraints of its various interactions explain why the PRD1 family has highly conserved coat proteins.

Does common architecture reveal a viral lineage spanning all three domains of life?

Our discovery that the major coat protein of bacteriophage PRD1 resembles that of human adenovirus raised the unexpected possibility that viruses infecting bacteria could be related by evolution to those infecting animal hosts. We first review the development of this idea. We then describe how we have used structure-based modeling to show that several other viruses with no detectable sequence similarity are likely to have coats constructed from similar proteins-the "double-barrel trimer." There is evidence that the group includes a diversity of viruses infecting very different hosts in all three domains of life: Eukarya; Bacteria; and Archaea that diverged billions of years ago. The current classification of viruses obscures such similarities. We propose that the occurrence of a double-barrel trimer coat protein in an icosahedral dsDNA virus with large facets, irrespective of its host, is a very strong indicator of its membership in a lineage of viruses with a common ancestor.

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state.

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.