Biomedical subjects

R Ficner

Publications and source records attributed to R Ficner.

At least 19 recordsLinked to original sources

PubMed2001

A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase.

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.

Binding Sites↗

PubMed2001

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) catalyses the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralisation of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. The enzyme was found to be functional towards a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also catalyses the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. The 3alpha-HSD/CR gene (hsdA) is 774 base pairs long and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed 3alpha-HSD/CR as a new member of the short-chain dehydrogenase/reductase (SDR) superfamily. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein indicating a dimeric nature of 3alpha-HSD/CR. The protein was crystallised and the structure solved by X-ray analysis. The crystal structure reveals one homodimer per asymmetric unit, thereby verifying its dimeric nature. Dimerisation takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far, this type of intermolecular contact has exclusively been observed in homotetrameric SDRs, but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain, which is missing in all other SDRs of known structure. The promoter domain was localised within the 93 bp region upstream of hsdA and the transcriptional start site was identified at 28 bp upstream of the translation start site. Interestingly, hsdA expression was found to be under negative control by two repressor proteins, the genes of which were found in opposite direction downstream or overlapping with hsdA. Based on our results, we propose that induction of hsdA expression in C. testosteroni by steroids actually appears to be a de-repression by preventing the binding of repressor proteins to regulatory regions.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family.

The crystal structure of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3alpha-HSDH) as well as the structure of its binary complex with NAD(+) have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSDH by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Crystal structure of the human U4/U6 small nuclear ribonucleoprotein particle-specific SnuCyp-20, a nuclear cyclophilin.

The cyclophilin SnuCyp-20 is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle involved in the nuclear splicing of pre-mRNA. It stably associates with the U4/U6-60kD and -90kD proteins, the human orthologues of the Saccharomyces cerevisiae Prp4 and Prp3 splicing factors. We have determined the crystal structure of SnuCyp-20 at 2.0-A resolution by molecular replacement. The structure of SnuCyp-20 closely resembles that of human cyclophilin A (hCypA). In particular, the catalytic centers of SnuCyp-20 and hCypA superimpose perfectly, which is reflected by the observed peptidyl-prolyl-cis/trans-isomerase activity of SnuCyp-20. The surface properties of both proteins, however, differ significantly. Apart from seven additional amino-terminal residues, the insertion of five amino acids in the loop alpha1-beta3 and of one amino acid in the loop alpha2-beta8 changes the conformations of both loops. The enlarged loop alpha1-beta3 is involved in the formation of a wide cleft with predominantly hydrophobic character. We propose that this enlarged loop is required for the interaction with the U4/U6-60kD protein.

Amino Acid Sequence↗

PubMed2000

Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment.

We have determined the crystal structure of a spliceosomal RNP complex comprising the 15.5kD protein of the human U4/U6.U5 tri-snRNP and the 5' stem-loop of U4 snRNA. The protein interacts almost exclusively with a purine-rich (5+2) internal loop within the 5' stem-loop, giving an unusual RNA fold characterized by two tandem sheared G-A base pairs, a high degree of purine stacking, and the accommodation of a single RNA base, rotated out of the RNA chain, in a pocket of the protein. Apart from yielding the structure of an important entity in the pre-mRNA splicing apparatus, this work also implies a model for the complex of the 15.5kD protein with box C/D snoRNAs. It additionally suggests a general recognition principle in a novel family of RNA binding proteins.

Amino Acid Sequence↗

PubMed2000

Screening orthologs as an important variable in crystallization: preliminary X-ray diffraction studies of the tRNA-modifying enzyme S-adenosyl-methionine:tRNA ribosyl transferase/isomerase.

The genes encoding the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging-drop vapour-diffusion method, crystals suitable for X-ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit-cell parameters a = b = 100.7, c = 150.9 A. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 A could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments.

Bacteria↗

PubMed1999

Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily.

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

Amino Acid Sequence↗

PubMed1999

Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15 kD protein.

The U5 small ribonucleoprotein particle (snRNP) contains various proteins involved in catalytic activities mediating conformational rearrangements of the spliceosome. We have isolated and characterized the evolutionarily highly conserved human U5 snRNP-specific protein U5-15kD. The crystal structure of U5-15kD determined at 1.4 A resolution revealed a thioredoxin-like fold and represents the first structure of a U5 snRNP-specific protein known so far. With respect to human thioredoxin the U5-15kD protein contains 37 additional residues causing structural changes which most likely form putative binding sites for other spliceosomal proteins or RNA. Moreover, a novel intramolecular disulfide bond replaces the canonical one found in the thioredoxin family. Even though U5-15kD appears to lack protein disulfide isomerase activity, it is strictly required for pre-mRNA splicing in vivo as we demonstrate by genetic depletion of its ortholog in Saccharomyces cerevisiae. Our data suggest that the previously reported involvement of its Schizosaccharomyces pombe ortholog Dim1p in cell cycle regulation is a consequence of its essential role in pre-mRNA splicing.

Amino Acid Sequence↗

PubMed1999

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.

Amino Acid Sequence↗

PubMed1999

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase to elucidate the role of serine 103 for enzymatic activity.

The tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041-7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction.

Crystallography↗

PubMed1999

Overproduction, purification, crystallization and preliminary x-ray diffraction studies of the human spliceosomal protein U5-15kD.

The gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding. The crystals belong to the orthorhombic space group P21212 with a = 62.3, b = 65.7, c = 37.1 A. They diffract to at least 3.0 A and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection.

Cloning, Molecular↗

PubMed1999

Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases.

The Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin. It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active holo-forms by 4'-phosphopantetheinylation. The B. subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized. Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant. The crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = b = 65.3, c = 150.5 A. They diffract beyond 2.8 A and contain one molecule in the asymmetric unit.

Bacillus subtilis↗

PubMed1997

Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori.

The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli. The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway. This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells. The predicted amino acid sequence of the H. pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes. The single gene on a plasmid was biologically active in E. coli. It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin. Two other reading frames were found adjacent to the aroB gene. The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity). The function of the tgt gene was confirmed by heterologous complementation. The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E. coli. The presence of the aroB gene and the putative tgt homologue in unrelated H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.

Amino Acid Sequence↗

PubMed1996

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.

Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.

Aspartic Acid↗

PubMed1996

Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase.

Based on the recently solved three-dimensional structure of pterin-4a-carbinolamine dehydratase from rat/human liver the involvement of the proposed active-site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site-directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57-->Ala mutant and the His61-->Ala, His62-->Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydropterin were significantly increased for binding to the Glu57-->Ala, His61-->Ala, His62-->Ala single mutants and the His61-->Ala, His62-->Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His61.

Amino Acid Sequence↗

PubMed1996

Structure and function of PCD/DCoH, an enzyme with regulatory properties.

The bifunctional protein PCD/DCoH is both an enzyme involved in the phenylalanine hydroxylation system and a transcription coactivator forming a 2:2 heterotetrameric complex with the nuclear transcription factor HNF1. The discovery of a bacterial homologue and the expression pattern during Xenopus embryogenesis suggest a regulatory function not only restricted to HNF1. The crystal structures of the tetrameric rat and the dimeric bacterial PCD/DCoH have led to the proposal of substrate and HNF1 binding sites. The saddle-shaped beta-sheet surfaces of the DCoH dimers likely represent binding sites for as yet unknown macromolecular interaction partners. Possible mechanisms for DCoH-induced transcriptional regulation are discussed in the light of the three-dimensional structures.

Amino Acid Sequence↗

PubMed1996

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

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.

Amino Acid Sequence↗

PubMed1996

Purification, crystallization, and preliminary x-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis.

The tRNA modifying enzyme tRNA-gnanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the quenine precursor 7-aminomethyl-7-deazagnanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for X-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 A, b = 65.1 A, c = 71.9 A, and beta = 97.5 degrees and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 A resolution.

Chromatography, Ion Exchange↗