Biomedical subjects
Publications and source records attributed to A Bacher.
The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.
The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.
Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.
A gene located at 443 kilobases on the Escherichia coli chromosome (subsequently designated ribE) was expressed in a recombinant E. coli strain and was shown to code for the enzyme 6, 7-dimethyl-8-ribityllumazine synthase. The recombinant enzyme was purified to homogeneity. The protein is an icosahedral capsid of 60 subunits with a mass of about 1 MDa as shown by hydrodynamic studies and by electron microscopy. In contrast to the icosahedral lumazine synthase-riboflavin synthase complex of Bacillus subtilis, the lumazine synthase of E. coli is not physically associated with another enzyme of the riboflavin pathway, and the core of the icosahedral capsid is empty. The RIB4 gene of Saccharomyces cerevisiae was also expressed to a high level (about 40% of cellular protein) in E. coli. The recombinant protein is a pentamer of 90 kDa. An insertion of 4 amino acids into helix alpha4 is likely to hinder the formation of an icosahedral capsid by the yeast protein. The kinetic properties of lumazine synthase of E. coli, B. subtilis, and S. cerevisiae are similar.
The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a mass of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein. The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30% of cellular protein. The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol.mg-1.h-1. The protein sediments at a velocity of S20 = 3.8 S. Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C-terminal and N-terminal parts suggesting two structurally similar folding domains.
The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.
The binding of small ligands to symmetrical oligomeric proteins may lead to a number of different partially ligated intermediates but should finally yield a symmetrical fully ligated enzyme/ligand complex. In the case of the trimeric protein, riboflavin synthase, some ligands form an unexpected protein/ligand complex, even in the presence of a large excess of ligand. Three different bound forms were observed by 19F NMR spectroscopy, and Scatchard-type analysis suggested binding sites of similar affinities. NOESY analysis of the kinetic network revealed that the three bound states exchange with free ligand, but not with each other, thus suggesting that the trimeric enzyme could be asymmetrical. This information permits appropriate precautions to be taken during X-ray structure analysis of riboflavin synthase, which is in progress. Quantitative analysis of the NOESY spectra yielded different rate constants for the different binding sites. For comparison, the monomeric lumazine protein was investigated as an example of a case with simple two-site exchange. For such systems, all kinetic parameters including kon and the dissociation constant can be determined from the NOESY spectrum. The data show that NMR spectroscopy can produce qualitative and quantitative information in cases of nonequivalent binding sites in oligomeric proteins if isolated NMR signals of the different forms can be observed. The technique is not limited to 19F as reporter nucleus.
A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524].
We have identified a genomic clone containing the 5' regulatory region of the gene GTP-CH encoding human GTP cyclohydrolase I. The transcription start point (tsp) was mapped by 5'-rapid amplification of cDNA ends (5'-RACE). The 2.6-kb region upstream from the tsp showed promoter activity when ligated upstream from a reporter gene. The truncation of approximately 2 kb of the promoter did not change expression activity, while a further removal of 243 bp halved the activity. The promoter contains CCAAT and TATA boxes. The GC-rich region close to the tsp, which contains several putative Sp1-responsive elements, is required for maximum promoter activity. Interferon-gamma treatment of B-cells transfected with reporter constructs had no influence on the expression activity.
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BACKGROUND: Anaesthesia induction and deep anaesthesia may be accompanied by a considerable haemodynamic depression, especially in patients suffering from cardiovascular diseases. A decrease in cardiac index (CI) leads to a concomitant decrease in oxygen transport (DO2I). We examined whether these changes in haemodynamic performance and oxygenation can cause an oxygen debt and anaerobic metabolism. METHODS: DO2I, oxygen uptake (VO2I), oxygen extraction ratio (O2ER) and plasma lactate were analysed at 9 pre-defined study stages during anesthesia induction, deep anaesthesia prior to surgery and during surgery in 65 patients (ASA 3) undergoing elective vascular surgery. Polynomials of increasing order were fitted to the data for the determination of the critical value of oxygen transport from the inflection point of the regression curve. RESULTS: CI, heart rate, mean arterial pressure and DO2I decreased significantly after anaesthesia induction and during deep anaesthesia. VO2I showed an almost parallel change during the study period so that O2ER remained nearly constant. Plasma lactate did not exceed the physiological range in any patient and a critical value of DO2I could not be detected because a linear regression always provided the best fit for the data. CONCLUSIONS: We conclude that in patients suffering from a substantial cardiovascular disease systemic oxygenation is not impaired by considerable haemodynamic changes induced by general anaesthesia. This fact can be explained by the parallel decrease in oxygen demand, expressed by the decrease in VO2I.
GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.
The lumazine synthase/riboflavin synthase of Bacillus subtilis is a bifunctional enzyme complex catalysing the formation of riboflavin from 5-amino-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and L-3,4-dihydroxy-2-butanone-4-phosphate via 6,7-dimethyl-8-ribityllumazine. The complex is composed of 3 alpha (riboflavin synthase) subunits and 60 beta (lumazine synthase) subunits and has a relative mass of 1 MDa. The 60 beta subunits are arranged in an icosahedral capsid enclosing the alpha trimer in the central core. The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 A resolution with a crystallographic R-factor of 0.33. Hollow, icosahedral capsids consisting of 60 beta subunits can be obtained by inhibitor-driven renaturation of isolated beta subunits. They catalyse the formation of 6,7-dimethyl-8-ribityllumazine at the same rate as the native alpha 3 beta 60 complex and can be crystallised in two different hexagonal and one monoclinic form. Crystallographic intensity data of the monoclinic crystals to a resolution of 2.4 A were obtained using synchrotron radiation and an image plate detector system. The orientation of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 A Patterson map. Phases were calculated from the model of the alpha 3 beta 60 protein and were extended by cyclic averaging exploring the 30-fold redundancy of the electron density. The 2.4 A map allowed us to refine the existing atomic model of lumazine synthase. The refined model includes 154 amino acid residues, one inhibitor molecule, 58 water molecules and one phosphate ion. Applying non-crystallographic-symmetry restraints the crystallographic R-factor is 16.7% for 100,092 reflections between 10 and 2.4 A. The chain folding of the beta subunits is closely similar to the native alpha 3 beta 60 enzyme. The lumazine synthase bears resemblance to the sugar binding proteins. The significantly higher resolution compared to the alpha 3 beta 60 structure determination allows a detailed description of the substrate analogue binding site. The environment of the 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione inhibitor is particularly rigid, and the chain segments involved in forming the active site are highly conserved for lumazine synthases of different species. A residual density feature in the final map is interpreted as a bound phosphate which mimics the binding of the second substrate. We discuss the reaction mechanism on this structural basis.
A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 A, b = 210.4 A, c = 71.8 A, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 A electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 A and a thickness of 65 A. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.