Biomedical subjects
L R Olsen
Publications and source records attributed to L R Olsen.
Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites.
N-Acetylglucosamine-1-PO(4) uridyltransferase (GlmU) is a trimeric bifunctional enzyme that catalyzes the last two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc. The X-ray crystal structure of Escherichia coli GlmU in complex with UDP-GlcNAc and CoA has been determined to 2.1 A resolution and reveals a two-domain architecture that is responsible for these two reactions. The C-terminal domain is responsible for the CoA-dependent acetylation of Glc-1-PO(4) to GlcNAc-1-PO(4) and displays the longest left-handed parallel beta-helix observed to date. The acetyltransferase active site defined by the binding site for CoA makes use of residues from all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO(4) acetyl acceptor. The N-terminal domain catalyzes uridyl transfer from UTP to GlcNAc-1-PO(4) to form the final products UDP-GlcNAc and pyrophosphate. This domain is composed of a central seven-stranded beta-sheet surrounded by six alpha-helices in a Rossmann fold-like topology. A Co(2+) ion binds to just one of the two independent pyrophosphorylase active sites present in the crystals studied here, each of which nonetheless binds UDP-GlcNAc. The conformational changes of the enzyme and sugar nucleotide that accompany metal binding may provide a window into the structural dynamics that accompany catalysis.
Purification, crystallization and preliminary X-ray data for Escherichia coli GlmU: a bifunctional acetyltransferase/uridyltransferase.
Crystals of Escherichia coli GlmU, a bifunctional enzyme catalyzing the acetylation of glucosamine-1-phosphate and uridylylation of N-acetylglucosamine-1-phosphate to produce UDP-GlcNAc, have been prepared in complex with coenzyme A and UDP-GlcNAc. These crystals belong to space group R32, with unit-cell parameters a = 104.5, c = 648.2 A, diffract to at least 2.1 A resolution and may contain two subunits of the trimeric enzyme per asymmetric unit.
[Logbook. A possible method].
The European board of psychiatry recommends the use of a logbook and checklist. There has been discussion about the extent of registration--and the resources involved. We present a simple method of registering supervision. Trainees make a daily report of the amount of supervision received. The trial period was divided into two sections: March, April and May 1997; (training intensive) and June, July and August 1997; (holiday season). The trainees responded well to the logbook, the answering rate was 94.2% in the first period, 60.9% in the holiday season. Calculations based on the first period show that trainees receive supervision during 9.8% of their working hours (range 4.8-16.0). Twenty-nine percent of supervision is of the direct variety, where both trainer and trainee are present. This variety suffers during holiday season. This type of logbook provides an opportunity to be directed in training activities, with a minimum of resources involved.
Insertions into the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase reveal a role for fingers subdomain in processive polymerization.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer. Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.
X-ray crystallographic studies of unique cross-linked lattices between four isomeric biantennary oligosaccharides and soybean agglutinin.
Soybean agglutinin (SBA) (Glycine max) is a tetrameric GalNAc/Gal-specific lectin which forms unique cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta et al. (1994) Biochemistry 33, 7495-7504]. We recently reported the X-ray crystal structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen [Dessen et al. (1995) Biochemistry 34, 4933-4942]. In order to determine the molecular basis of different carbohydrate-lectin cross-linked lattices, a comparison has been made of the X-ray crystallographic structures of SBA cross-linked with four isomeric analogs of the biantennary blood group I carbohydrate antigen. The four pentasaccharides possess the common structure of (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3. The beta-LacNAc moieties in the four carbohydrates are linked to the 2,3-, 2,4-, 3,6-, and 2,6-positions of the core Gal residue(s), respectively. The structures of all four complexes have been refined to approximately 2.4-2.8 A. Noncovalent lattice formation in all four complexes is promoted uniquely by the bridging action of the two arms of each bivalent carbohydrate. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharides at identical sites in each monomer, with the sugar(s) cross-linking to a symmetry-related neighbor molecule. While the 2,4-, 3,6-, and 2,6-pentasaccharide complexes possess a common P6422 space group, their unit cell dimensions differ. The 2, 3-pentasaccharide cross-linked complex, on the other hand, possesses the space group I4122. Thus, all four complexes are crystallographically distinct. The four cross-linking carbohydrates are in similar conformations, possessing a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry in each lattice. In the case of the 3,6- and 2,6-pentasaccharides, the symmetry of their cross-linked lattices requires different rotamer orientations about their beta(1,6) glycosidic bonds. The results demonstrate that crystal packing interactions are the molecular basis for the formation of distinct cross-linked lattices between SBA and four isomeric pentasaccharides. The present findings are discussed in terms of lectins forming unique cross-linked complexes with glycoconjugate receptors in biological systems.
The nucleoside analog-resistant E89G mutant of human immunodeficiency virus type 1 reverse transcriptase displays a broader cross-resistance that extends to nonnucleoside inhibitors.
The alteration of a glutamic acid (E) to a glycine (G) amino acid residue at position 89 (E89G alteration) in the human immunodeficiency virus type 1 reverse transcriptase confers decreased susceptibility to several nucleoside analog inhibitors. Because the nonnucleoside inhibitor-binding pocket is adjacent to the deoxynucleoside triphosphate substrate-binding site, the impact of the E89G reverse transcriptase has decreased susceptibility to TIBO R82150, nevirapine, and to a lesser extent, delavirdine. Human immunodeficiency viruses bearing the same mutation displayed decreased susceptibility to inhibition by these compounds in a cell culture virus replication assay.
Circular dichroism and Fourier transform infrared spectroscopic studies on the secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate carboxykinases.
The secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate (PEP) carboxykinases was quantitatively examined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies. From CD analyses, values of 24% alpha-helix and 38% beta-sheet were obtained for the E. coli enzyme, while the corresponding values for the S. cerevisiae PEP carboxykinase were 20% and 36%. Analysis of the amide I' infrared band indicated 20% alpha-helix and 36% beta-sheet for the S. cerevisiae enzyme, while for the E. coli protein values of 40% beta-sheet and between 9 and 36% alpha-helix could be inferred. It is concluded that the bacterial enzyme has more secondary structure elements than the yeast protein. No alteration of the CD or FTIR spectra was detected upon substrate or metal ion binding to any enzyme.
The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase.
The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.