Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position- dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.      

DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides

A series of hairpin pyrrole/imidazole polyamides linked to a Hoechst 33258 (Ht) analogue (5-7) were synthesized on solid-phase by adopting an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. The dsDNA binding properties of Ht-polyamides 5-7 were determined by thermal denaturation experiments. Hairpin Ht-polyamides 5-7 bound to dsDNA sequences 16 and 18 show DeltaTm values that are 14-18 degrees higher than linear Ht-polyamides bound to the same sequences. All three Ht-polyamides were found to be selective for their 9-bp match dsDNA sequences, supporting a relative stronger interaction of an Im/Py anti-parallel dimer with an appropriately positioned G/Cbp rather than sequences containing only A/Tbps. In addition, Ht-polyamides 5 and 7 showed a 20-fold preference for a properly placed G/Cbp over a C/Gbp, while 6 showed a 10-fold preference.     

Complexation of single strand telomere and telomerase RNA template polyanions by deoxyribonucleic guanidine (DNG) polycations: plausible anticancer agents

Cancerous cell immortality is due to relatively high concentrations of telomerase enzyme which maintains telomere sequence during cell division. Deoxyribonucleic guanidine (DNG) is a positively charged DNA analog in which guanidine replaces the phosphordiester linkage of DNA. Mixed sequences of DNG and DNA oligonucleotides are referred to as chimera. Complexation of DNG and chimeric polycations with the complementary negatively charged non-coding telomere single strand d(5'-TTAGGG-3')(n) and the 11-base telomeric RNA template (5'-CUAACCCUAAC-3') in the active site of telomerase has been studied. Calculated by ensemble sampling simulations in GBMV solvent model, we found that binding of complementary DNG hexamer with telomere is favored over that of DNA-telomere by approximately 10(6)-fold and binding of chimera hexamer is favored by approximately 10(4)-fold. Binding of complementary DNG with telomeric RNA is favored by 43 kcal/mol over telomere substrate binding with telomeric RNA.     

Multiple substrate binding states and chiral recognition in cofactor-independent glutamate racemase: a molecular dynamics study

Glutamate racemase (MurI) catalyzes the racemization of glutamate; two cysteine residues serve as catalytic acid and base. On the basis of the crystal structure of MurI from the hyperthermophilic bacterium Aquifex pyrophilus, we performed molecular dynamics (MD) simulations of six different systems to investigate stereochemistry, substrate ligation, and active site protonation state. The catalytic competence of individual systems was assessed by the abundance of reactive conformers. Only systems in which Cys70 is poised to deprotonate d-Glu were found to be catalytically competent (idem Cys178/l-Glu), in agreement with the experimentally observed stereochemistry of Lactobacillus fermentii MurI [Tanner, M. E. et al. (1993) Biochemistry 32, 3998-4006]. Only systems in which the alpha-amino group of l/d-Glu and the imidazole moiety of His are deprotonated are catalytically competent. The active site of MurI displays an unusual flexibility in substrate ligation, and several transitions between stable binding patterns were observed. In catalytically competent binding states, the conserved threonine residues 72, 114, and 117 ligate the alpha-carboxylate of Glu and the Asn71 amides ligate the alpha-amino group of Glu, whereas the delta-carboxylate of Glu is steered by electrostatic repulsion from the Asp7 and Glu147 side chain carboxylates. A network of hydrogen bonds controls the positioning of each thiol/thiolate. In what we term substrate flipping, Glu suddenly rotates into a binding pattern that resembles the post-racemization state of the other enantiomer, i.e., each enantiomer can be bound in two distinct states. Substrate flipping and unfavorable substrate binding successively trigger dissociation of the substrate, accompanied by an opening of the active site channel. We explain how the weak binding of Glu contributes to catalysis and suggest a mechanism by which binding mismatches are propagated into an opening of the active site.     

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.     

Generation and evaluation of putative neuroregenerative drugs. Part 1: virtual point mutations to the polyketide rapamycin

This paper explores the use of computational methods to direct engineered biosynthesis based on the desired properties of the target compounds. The immunosuppressive properties of rapamycin are a result of the formation of the complex FKBP12-rapamycin-FRAP. Neuroregenerative properties are exhibited by the complex or complexes of rapamycin with FKBP proteins. Our objective has been to design biosynthetically available analogues of rapamycin that bind tightly to FKBP12 but not to FRAP. This has been carried out by successive single ketide deletions from the effector domain of rapamycin. The approach described here has yielded modified rapamycin analogues (RP2 and RP3) as targets for biosynthesis by modified polyketide synthases. RP2 and RP3 have an identical binding affinity (linear interaction energy calculation) to FKBP12 as rapamycin but little or no affinity for binding to FRAP.