no abstract available
MARCY HERNICK and Carol A. Fierke
University of Michigan, Department of Chemistry, Ann Arbor, MI 48109
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine, the committed step in the biosynthesis of Lipid A. The catalytic mechanism of this enzyme has been investigated using a combination of site-directed mutagenesis and kinetics experiments. The effect of the E78A, H265A, E78A/H265A and D246A active site mutations on the steady-state parameters (KM, kcat, kcat/KM) were determined and the results indicate that E78, H265 and D246 are all important for catalytic activity. Furthermore, the pH-dependence of the LpxC-catalyzed reaction under steady-state conditions (kcat/KM) indicates that there are at least two ionizations that are important for catalytic activity. Mutagenesis and metal-substitution experiments were used to assign the observed pKa's to reflect the ionization of Glu78 and the zinc bound water molecule. Additionally, a kinetic solvent isotope effect of two is observed for WT LpxC, consistent with participation of a general-base catalyst in the catalytic mechanism. These data, in the context of the x-ray crystal structure of A. aeolicus LpxC, suggest a unique general- acid-base catalyst pair mechanism that is distinct from the mechanism of the zinc metalloproteases.
(Supported by NIH grant GM 40602)
BRANDON J. LAMARCHE, Alexander K. Showalter, and Ming-Daw Tsai
The Ohio State University, Department of Chemistry, Columbus, OH 43210
By continually varying their surface antigens many pathogens are able to evade the immune response of their host. A mechanistic understanding of these morphing processes is critical since their inhibition, if not lethal to the pathogen, could potentially slow antigen variation sufficiently for the host immune system to "catch" its prey. Though high mutation frequencies are often attributable to low fidelity DNA replication, in the work described here we have attempted to ascertain whether sequence diversification might also be effected by an error prone base excision repair (BER) system.
The African Swine Fever Virus (ASFV) is a large double stranded DNA virus that replicates and assembles in the cytoplasm of porcine macrophage cells. Consistent with its intracellular location, ASFV codes for enzymes involved in both the replication and repair of DNA. ASFV is hypervariable, and in particular shows an abundance of point mutations among its different isolates.
We previously hypothesized that the highly error-prone DNA polymerase X (Pol X) from the African Swine Fever Virus (ASFV) might contribute to ASFV hypermutation via mutagenic BER. Herein we provide enzymatic evidence accounting for all requisite ASFV BER activities - including a unique ASFV enzyme that acts both as a DNA lyase and a low fidelity DNA ligase and demonstrate that the fidelity-determining components of this system are error prone. These results are the first enzymatic evidence for a virally encoded BER pathway, and constitute the first evidence for an error prone BER system.
We suggest that, similar to emerging models of immunoglobulin hypermutation, low fidelity DNA repair - initiated at sites of base modification - is a feasible mechanism for promoting sequence diversification in ASFV.
S. R. DAS and J. A. Piccirilli,
Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology and Chemistry, The University of Chicago, Chicago, IL 60637
While protein enzymes possess numerous functional groups that permit optimal catalytic activity, RNA enzymes (ribozymes), which have limited functionality, can also catalyze chemistry with similar efficiency. RNA's catalytic prowess was thought to arise from metal ions organized by the scaffold of the RNA motif. However, the view that RNA nucleobases may provide catalytic capability is garnering recognition; even ribosomal peptide synthesis is proposed to be nucleobase mediated. In all known naturally occurring small ribozymes, activity in the absence of divalent metal ions posits possible nucleobase involvement in catalysis. In the human hepatitis delta virus (HDV) ribozyme, a specific cytosine residue observed at the active site in the crystal structure was proposed to provide the catalytic ability to cleave a phosphodiester bond through proton transfer. However, the role of this putative catalytic residue in proton transfer is ambiguous. We have developed a method to resolve the inherent ambiguity in kinetic analysis of general acid-base catalysis. We establish through chemical manipulation and biochemical analyses a direct link between a single atom on the substrate cleavage site and a single atom of the HDV ribozyme active site cytosine. Thereby we elucidate the role of the cytosine in proton transfer that potentiates catalysis. Our chemical dissection of the delta ribozyme active site residue reveals the efficacy of RNA nucleobases in biocatalysis.
BARBARA GOLDEN
Purdue University, Department of Biochemistry, West Lafayette, IN 47907.
Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-12 intron from phage Twort at 3.6 Å resolution bound to a four nucleotide product RNA. In addition to the three conserved domains characteristic of all group I introns, the Twort intron, like most bacteriophage introns, possesses peripheral insertions that form a ring that completely envelops the active site where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2'-hydroxyl of the guanosine substrate in organization of the active site for catalysis.
CHRISTINE A. HRYCYNA
Purdue University, Department of Chemistry, West Lafayette, IN 47907.
Numerous proteins, including Ras, nuclear lamins, and yeast a-factor, contain a C-terminal CaaX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids (-aaX), and a-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenyl carboxyl methyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste 14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotic organisms. Notably, Ste14p, like all Icmt's, has multiple membrane spanning domains, which present a significant challenge to its purification in an active form. In this study, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary to promote the carboxylmethylation of isoprenylated substrates. Optimal solubilization and retention of Ste14p activity occurred with the detergent ß-D- dodecylmaltoside (DDM), among the extensive panel of detergents that were screened. The activity of Ste14p could be further optimized (~3-fold) upon reconstitution of purified detergent-solubilized Ste14p into liposomes. Our expression and purification scheme generates milligram quantities of pure and active Ste 14p, which is highly stable under a variety of conditions. Evidence that metals ions are required for activity of Ste14p is presented. We also show that in addition to catalyzing methylation of a model substrate (AFC), purified Ste14p can methylate a known in vivo substrate, Ras2p. These results pave the way for biophysical and biochemical characterization of pure Ste 14p, as well as determination of its three-dimensional structure.
Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of function, making Icmt an attractive and novel anti-cancer target. Isoprenoid- modified analogs of the minimal Icmt substrate N-acetyl farnesyl cysteine were synthesized and found to vary widely in their ability to act as substrates, confirming that the isoprenoid moiety is a key substrate recognition element for Icmt. A farnesyl isobutenyl derivative and a farnesyl isobutenyl biphenyl derivative are competitive inhibitors of overexpressed yeast Icmt (K;= 17.1 ± 1.7 μM and 11.7 ± 1.0 μM, respectively) and also block the methylesterification of the physiological substrate, Ras2p.
Amy Roth', Beimeng Sun1, Aaron Bailey2, Jason Kuchar3, William Green4, Brett Phinney3, John Yates, III2, NICHOLAS DAVIS'; '
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI; 'Department of Cell Biology, The Scripps Research Institute, La Jolla, CA; 3Department of Biochemistry, Michigan State University, East Lansing, MI; 'Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, IL.
Our understanding of protein S-acylation (palmitoylation) lags well behind that of prenylation and myristoylation, the two other lipidations that tether proteins to the cytoplasmic surfaces of membranes. The peptide motifs that specify and accept the palmitoyl modifications remain vague and first examples of the enzymes mediating this lipid modification, i.e. the protein acyl transferases (PATs), have been identified only recently. Our lab identified the yeast protein Akr1p as the PAT for two plasma membrane casein kinases, Yck1p and Yck2p, and another yeast group identified a complex of Erf2p and Erf4p as the PAT for the yeast Ras proteins, Ras1p and Ras2p. Akr1p localizes to the Golgi; Erf2p/Erf4p localizes to the ER. While lacking extended homology, Akr1p and Erf2p both are predicted polytopic membrane proteins and both contain the novel 51 residue-long, zinc finger-like DHHC cysteine-rich domain (DHHC- CRD). The DHHC-CRD sequence defines a diverse family of proteins distributed throughout the eukaryotic kingdom; yeast has seven DHHC proteins, while humans have 23. All are predicted to be polytopic membrane proteins and all have the DHHC-CRD similarly positioned within a predicted cytoplasmic loop. Our working hypothesis is that the DHHC protein family constitutes a family of PATs, the diversity of which may accommodate the diversity of proteins known to be palmitoylated. As a tool for linking palmitoylated proteins to their cognate PAT, we have a developed proteomic methodology that purifies and identifies palmitoylated proteins from complex protein extracts. The crux of the method is an in vitro exchange of biotinyl modifications for the protein acyl modifications. The biotinylated proteins are then purified and identified by tandem MS. Palmitoyl-proteome comparisons of wild-type and DHHC gene-deleted yeast should reveal the linkages of the PATs to their protein targets.
Julie A. Francois, Aaron E. Ransome, Charles Z. Constantine, Christopher P. Mill, Sasitorn Sivanuntakorn, Rena Goodman, Jeong Won Nam, and T. JOSEPH KAPPOCK
Department of Chemistry, Washington University in St. Louis, One Brookings Drive, Campus Box 1134, Saint Louis, MO 63130-4899
Bacteria living in harsh environments often have cell components adapted to have increased stability against one or more destabilizing conditions. Acetobacter aceti converts ethanol into acetic acid (vinegar) and is therefore highly acid-resistant: it can thrive with internal pH = 4. Acetic acid is particularly toxic because it easily permeates cells as uncharged acid pairs, poisoning susceptible organisms. Since A. aceti lives with a constant influx of acetic acid, it differs from acidophilic organisms that survive exposure to membrane-impermeant inorganic acids. Thus every enzyme in A. aceti should function in conditions hostile to most familiar cytoplasmic enzymes.
We purified several of these enzymes (citrate synthase, alanine racemase, and N3- carboxy-4-aminoimidazole ribonucleotide mutase) and found they are indeed unusually resistant to acid-mediated inactivation. Biophysical, enzymological, and crystal structure data have given insight into several features that might be employed by proteins resistant to acid-mediated destabilization.
This project was supported by grants from the Herman Frasch Foundation and the National Science Foundation.
LUSONG LUO, Jeffrey D Carson, Robert A Copeland
Department of Enzymology and Mechanistic Pharmacology, MMPD CEDD, GlaxoSmithKline, Collegeville, PA 19426.
Kinesin motor proteins are molecular machines that utilize the energy from ATP hydrolysis to transport cellular cargo along microtubules. Kinesins that play essential roles in the mechanics of mitosis are attractive targets for novel antimitotic cancer therapies. In this presentation, we show that the inhibition of KSP (kinesin spindle protein), the key kinesin protein involved in establishment of spindle bipolarity, by small molecule inhibitors can lead to mitotic arrest. We are going to review the detailed biochemical mechanisms of inhibition by the KSP inhibitors. In addition, the effect of ionic strength on the KSP inhibitors will be discussed.