pyrimidinones and queuine

There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60-70%) that is transcribed into non-coding RNA (Mattick [10]), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [(3)H] preQ(1) (a queuine precursor in eubacteria) clearly demonstrate preQ(1) incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ(1) incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.

Topics: Acrylic Resins; Electrophoresis, Agar Gel; Escherichia coli; Guanine; Isotope Labeling; Pyrimidinones; Pyrroles; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Transfer; Tritium

To thoroughly study the functional role of prokaryotic t-RNA-guanine-transglycosylases which are essential in the pathogenesis of shigellosis, novel efficient, high-yielding synthetic approaches for preQ(1) base, Q base, as well as for (ent)-Q base mainly employing cheap and readily available starting materials have been developed. Q base as well as (ent)-Q base are accessible starting from preQ(1) base via nucleophilic substitution reactions with appropriately decorated halocyclopentenyl synthons, prior to that prepared from naturally occurring carbohydrates.

Topics: Guanine; Molecular Structure; Pyrimidinones; Pyrroles

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.

Topics: Amino Acid Sequence; Escherichia coli; Evolution, Molecular; Guanine; Humans; Kinetics; Molecular Sequence Data; Mutation; Pentosyltransferases; Phylogeny; Pyrimidinones; Pyrroles; Sequence Homology, Amino Acid; Substrate Specificity

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modification of this initial lead.

Topics: Binding Sites; Computer Simulation; Crystallography, X-Ray; Databases as Topic; Drug Design; Dysentery, Bacillary; Enzyme Inhibitors; Guanine; Kinetics; Models, Molecular; Molecular Structure; Pentosyltransferases; Phthalazines; Pyrimidinones; Pyrroles; Shigella flexneri; Software; Static Electricity; Thermodynamics; Zymomonas

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.

Topics: Amino Acid Sequence; Anticodon; Catalysis; Crystallography, X-Ray; Guanine; Metalloproteins; Models, Molecular; Molecular Sequence Data; Nucleic Acid Precursors; Pentosyltransferases; Pyrimidinones; Pyrroles; Recombinant Proteins; RNA, Transfer; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Zinc; Zymomonas

A series of 5-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT [Hoops, G. C., Garcia, G. A., & Townsend, L. B. (1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23-28, 1992, Division of Medicinal Chemistry, Abstract 113]. The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-[14C]G34-tRNA. This study reveals that the tRNA-guanine transglycosylase from E. coli will tolerate a wide variety of substituents at the 5-position. The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis. A correlation between N7 pKa and Vmax suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate. Comparison of the Kis of 7-methyl-substituted competitive inhibitors to the Kms of their corresponding substrates suggests that some substrates (including preQ1) are kinetically "sticky" (i.e., Km is equivalent to Kd) and other substrates have Kms that reflect catalytic rates as well as binding.

Topics: Binding, Competitive; Enzyme Inhibitors; Escherichia coli; Guanine; Kinetics; Molecular Structure; Pentosyltransferases; Purines; Pyrimidinones; Pyrroles; RNA, Transfer; Structure-Activity Relationship; Substrate Specificity