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Glenn E Dale

Publications and source records attributed to Glenn E Dale.

10 recordsLinked to original sources
PubMed2006

Mapping the active site of Escherichia coli malonyl-CoA-acyl carrier protein transacylase (FabD) by protein crystallography.

Malonyl-CoA-acyl carrier protein transacylase (FabD; EC 2.3.1.39) is a key enzyme in the fatty-acid biosynthesis pathway of bacteria, catalyzing the transfer of a malonyl moiety from malonyl-CoA to holo acyl carrier protein (ACP), generating malonyl-ACP and free CoASH. Malonyl-ACP, which is the product of this reaction, is the key building block for de novo fatty-acid biosynthesis. Various binary complex structures of the Escherichia coli enzyme are presented, including that of the natural substrate malonyl-CoA, indicating the functional role of the highly conserved amino acids Gln11, Ser92, Arg117 and His201 and the stabilizing function of the preformed oxyanion hole during the enzymatic reaction. Based on the presented structural data, a possible new catalytic enzyme mechanism is discussed. The data obtained could be used in aiding the process of rational inhibitor design.

Acyl-Carrier Protein S-Malonyltransferase↗
PubMed2005

The crystal structure of E.coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation.

The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 A resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 A crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.

Aldose-Ketose Isomerases↗
PubMed2004

Crystal structures of Staphylococcusaureus methionine aminopeptidase complexed with keto heterocycle and aminoketone inhibitors reveal the formation of a tetrahedral intermediate.

High-resolution crystal structures of Staphylococcus aureus methionine aminopeptidase I in complex with various keto heterocycles and aminoketones were determined, and the intermolecular ligand interactions with the enzyme are reported. The compounds are effective inhibitors of the S. aureus enzyme because of the formation of an uncleavable tetrahedral intermediate upon binding. The electron densities unequivocally show the enzyme-catalyzed transition-state analogue mimicking that for amide bond hydrolysis of substrates.

Amines↗
PubMed2004

Structural analysis of neprilysin with various specific and potent inhibitors.

Neutral endopeptidase (NEP) is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor. Owing to the physiological importance of NEP in the modulation of nociceptive and pressor responses, there is considerable interest in inhibitors of this enzyme as novel analgesics and antihypertensive agents. Here, the crystal structures of the soluble extracellular domain of human NEP (residues 52-749) complexed with various potent and competitive inhibitors are described. The structures unambiguously reveal the binding mode of the different zinc-chelating groups and the subsite specificity of the enzyme.

Binding, Competitive↗
PubMed2003

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors.

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.

Amino Acid Sequence↗
PubMed2003

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD).

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.

Acyl-Carrier Protein S-Malonyltransferase↗
PubMed2003

High-resolution structure of human apo dipeptidyl peptidase IV/CD26 and its complex with 1-[([2-[(5-iodopyridin-2-yl)amino]-ethyl]amino)-acetyl]-2-cyano-(S)-pyrrolidine.

Dipeptidyl peptidase IV is a multifunctional type II transmembrane serine protease glycoprotein. The high-resolution crystal structure of the homodimeric human apo dipeptidyl peptidase IV has been determined at 1.9 A resolution. In addition, the structure of the binary complex with 1-[([2-[(5-iodopyridin-2-yl)amino]-ethyl]amino)-acetyl]-2-cyano-(S)-pyrrolidine has been solved, revealing the nature of the covalent interaction with the active-site serine.

Aminopyridines↗
PubMed2003

The protein as a variable in protein crystallization.

Strategies for growing protein crystals have for many years been essentially empirical, the protein, once purified to a certain homogeneity, being mixed with a selection of crystallization agents selected in a more or less trial-and-error fashion. Screening for the correct conditions has been made easier through automation and by the introduction of commercially available crystallization kits. Many parameters can be changed in these experiments, such as temperature, pH, and ionic strength, but perhaps the most important variable has been ignored, namely the protein. The crystallization properties of a protein vary greatly: some crystallize readily, whereas others have proven extremely difficult or even impossible to obtain in a crystalline state. The possibility of altering the intrinsic characteristics of a protein for crystallization has become a feasible strategy. Some historical perspectives and advances in this area will be reviewed.

Crystallization↗
PubMed2003

Specialized osmotic stress response systems involve multiple SigB-like sigma factors in Streptomyces coelicolor.

Whereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB-like genes in S. coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S. coelicolor. Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro. Its sigma subunit was identified as a product of the sigH gene, which is co-transcribed downstream of a putative antisigma factor gene (prsH). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis. Levels of two different sigH-encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post-translational regulatory elements such as PrsH. In addition, synthesis of three other SigH-like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated. This family of SigH-like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co-ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.

Bacterial Proteins↗