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Biomedical subjects

J Clardy

Publications and source records attributed to J Clardy.

At least 37 records · Page 2Linked to original sources
PubMed2000

Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid.

RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3. We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region. These genes were located on plasmid pa of R. etli CE3, which is self-transmissible between R. etli and A. tumefaciens.

Base Sequence↗
PubMed2000

Differences in medical students' empathy.

Medical students' Balanced Emotional Empathy Scale scores were compared by year, sex, and expressed specialty choice. Scores were lower for students choosing non-core specialties, and for M4 men students compared with M3 men students.

Analysis of Variance↗
PubMed2000

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Amino Acid Sequence↗
PubMed2000

Biological activity of guanacastepene, a novel diterpenoid antibiotic produced by an unidentified fungus CR115.

Fermentation extracts of culture CR115, an unknown plant endophyte originally isolated from Costa Rica, were found to be active against antibiotic-resistant bacteria. The metabolite responsible for activity was identified as a novel diterpenoid antibiotic guanacastepene (mol. wt. 374.47 and mol. formula C22H30O5). Mechanistic studies done in an E. coli imp strain suggested membrane damage as the primary mode of bactericidal action. This compound also lysed human RBCs and caused leakage of intracellular potassium from E. coli imp.

Anti-Bacterial Agents↗
PubMed1999

Synthesis, biological activity, and absolute stereochemical assignment of NPS 1392: a potent and stereoselective NMDA receptor antagonist.

The synthesis, biological activity, and single crystal X-ray structure of NPS 1392, (R)-(-)-3,3-bis(3-fluorophenyl)-2-methylpropan-1-amine (3a), a potent, stereoselective antagonist of the NMDA receptor, are described. The NMDA receptor selectively bound the levo isomer (3a) over its enantiomer (3b), which prompted a rigorous absolute configuration assignment. NPS 1392 has the R configuration based on the single-crystal X-ray diffraction analysis of the hydroiodide salt of NPS 1392. This compound is a potential neuroprotective agent for use in the treatment of ischemic stroke.

Animals↗
PubMed1999

The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease.

BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81. CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.

Amino Acid Sequence↗
PubMed1999

Structure of the PIN/LC8 dimer with a bound peptide.

The structure of the protein known both as neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), and also as the 8 kDa dynein light chain (LC8) has been solved by X-ray diffraction. Two PIN/LC8 monomers related by a two-fold axis form a rectangular dimer. Two pairs of alpha-helices cover opposite faces, and each pair of helices packs against a beta-sheet with five antiparallel beta-strands. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. A 13-residue peptide from nNOS is bound to the dimer in a deep hydrophobic groove as a sixth antiparallel beta-strand. The structure provides key insights into dimerization of and peptide binding by the multifunctional PIN/LC8 protein.

Amino Acid Sequence↗
PubMed1999

Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution.

The structure of the FKBP12-rapamycin-FRB ternary complex has now been refined at 2.2 A resolution. The cell-cycle arrest agent rapamycin binds FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin associated protein (FRAP) simultaneously, and the inhibition of FRAP is responsible for rapamycin's biological activity. The conformation of rapamycin in the ternary complex is very similar to that observed in the FKBP12-rapamycin binary complex, with an r.m.s. difference of only 0.30 A. However, a slight (9 degrees ) rotation repositions the FRB-binding face of rapamycin in the ternary complex. There are extensive rapamycin-protein interactions and relatively few interactions between the two protein partners FKBP12 and FRB, these interactions mainly involving residues in the 40s and 80s loops of FKBP12 and alpha1 and alpha4 of FRB. The high-resolution refinement has revealed the crucial role of several buried waters in the formation of the ternary complex.

Amino Acid Sequence↗
PubMed1999

ZmaR, a novel and widespread antibiotic resistance determinant that acetylates zwittermicin A.

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.

Acetylation↗
PubMed1998

Structure of human methionine aminopeptidase-2 complexed with fumagillin.

The fungal metabolite fumagillin suppresses the formation of new blood vessels, and a fumagillin analog is currently in clinical trials as an anticancer agent. The molecular target of fumagillin is methionine aminopeptidase-2 (MetAP-2). A 1.8 A resolution crystal structure of free and inhibited human MetAP-2 shows a covalent bond formed between a reactive epoxide of fumagillin and histidine-231 in the active site of MetAP-2. Extensive hydrophobic and water-mediated polar interactions with other parts of fumagillin provide additional affinity. Fumagillin-based drugs inhibit MetAP-2 but not MetAP-1, and the three-dimensional structure also indicates the likely determinants of this specificity. The structural basis for fumagillin's potency and specificity forms the starting point for structure-based drug design.

Amino Acid Sequence↗
PubMed1998

Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis.

Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.

Amino Acid Substitution↗
PubMed1998

Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins.

The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate.

Allosteric Regulation↗
PubMed1998

Chemical inducers of dimerization: the atomic structure of FKBP12-FK1012A-FKBP12.

A ligand that simultaneously binds two proteins must have two high affinity protein binding domains joined in a fashion that facilitates, or at least does not prevent, protein-protein interaction. Designing such ligands is challenging, and a high resolution X-ray structure of FKBP12-FK1012A-FKBP12 illustrates the subtleties of one successful design.

Dimerization↗