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   2013 - 2014 RESEARCH NEWS
 
Summaries of Recent Publications by HWI Scientists
 
Recent studies of dihydrofolate reductase

Much of Dr. Vivian Cody’s work is concerned with dihydrofolate reductase (DHFR), an enzyme that is a useful target for cancer chemotherapy as well as treatment of the type of pneumonia that often plagues AIDS patients.  Several of Cody’s recent papers cover various aspects of her DHFR-related work with collaborators at the Indiana University School of Medicine and Duquesne University.

The first paper reports not only the first kinetic data for a potent inhibitor (OAAG324) of dihydrofolate reductase from Pneumocystis jirovecii, Pneumocystis carinii and human enzyme sources, but also the first structural data of this inhibitor bound to three different active-site variants of human DHFR (PMID 23545530). A second paper describes the first kinetic data for variants of Pneumocystis jirovecii DHFR that have been observed in clinical isolates from patients with HIV and resistance to the drug trimethoprim (PMID 23896474). A third paper reports the crystal structures of a series of potent piritrexim analogues that are more selective for Pneumocystis jirovecii DHFR than for human or Pneumocystis carinii DHFR (PMID 23627352).

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Homology model of Pneumocystis jirovecii DHFR (green) with cofactor (yellow) and trimethoprim (cyan) highlighting the locations of 22 individual sites where amino-acid substitutions have been observed in clinical samples from patients with HIV/AIDS. [Learn more]

Substrate binding and activation of fatty acid α-dioxygenase

Fatty acid α-dioxygenases (α-DOX) are heme-containing proteins that generate potent lipid mediators, termed oxylipins, via the oxygenation of 14-20 carbon fatty acid substrates. α-DOX are generally found in plants and fungi, where they are up regulated during the host defense response against pathogen attack. These enzymes represent a subfamily within the cyclooxygenase-peroxidase superfamily, which include the mammalian peroxidases and cyclooxygenases.

Dr. Michael Malkowski and members of his laboratory have determined the X-ray crystal structures of α-DOX in complex with hydrogen peroxide and with the fatty acid substrate palmitic acid. These structures provide a detailed molecular view of the changes associated with the binding of substrate within the active-site cleft and the activation of catalysis by hydrogen peroxide. [Learn more]

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The structural basis underlying the activation of α-DOX by hydrogen peroxide.

 
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Studying molecules that allow bacteria to acquire iron

Many bacteria produce siderophores, molecules that they use to acquire iron when growing in low-iron environments. Bacteria require iron to live and devising methods to prevent iron acquisition by means of siderophores could help fight infections.

Many siderophores are derived from peptides and are produced by an interesting family of multi-domain proteins called non-ribosomal peptide synthetases (NRPS). These NRPS proteins catalyze specific reactions to link amino acid building blocks to produce the final peptide molecules.

Dr. Andrew Gulick and members of his laboratory are investigating the function of these multidomain NRPS enzymes through the tools of chemical and structural biology. They have used specifically designed inhibitors that allow them to trap the protein mid-reaction and capture the transient interaction of these protein domains. These studies are allowing them to use crystallography to provide individual snapshots of these dynamic reactions. Eventually, this basic research may lead to methods for inhibiting NRPS enzymes and siderophore synthesis. [Learn more]

The crystal structure of the NRPS adenylation domain from EntE (shown in blue) fused with the carrier protein domain from EntB (ribbon representation shown in red).

 
 
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Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant

Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed.

Dr. Edward Snell and his collaborators provide a new look at this issue with their report of the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data. [Learn more]

 

Metal-ion coordination at the active site of cyclic D-glucose-bound E186Q xylose isomerase.

 
Cyclooxygenase catalysis and inhibition in lipid bilayer nanodiscs

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid to generate potent lipid signaling molecules collectively known as prostaglandins (PGs). Changes in COX-mediated PG biosynthesis are associated with various disease pathologies, including inflammation, cardiovascular disease, and cancer. COX-1 and COX-2 are the targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). These compounds are some of the most heavily utilized drugs in the world, used to decrease acute and chronic inflammation, protect against adverse cardiovascular events, and reduce the risk of developing certain cancers. COX catalysis and inhibition takes place in the context of a membrane environment. As a consequence, maintenance of these enzymes in a stable and active form in solution for functional and structural characterization has traditionally required the presence of detergents. Nanodiscs, comprised of a small circular patch of lipid bilayer that is rendered soluble by two amphipathic α-helical membrane scaffold proteins that encircle the circumference of the bilayer technology, have proven to be a valuable tool in the study of membrane protein structure and function.

The Malkowski laboratory has successfully applied nanodisc technology to reconstitute COX-2 into nanodiscs and subsequently characterized nanodisc-reconstituted COX-2 catalysis and inhibition. Nanodisc-reconstituted COX-2 exhibited characteristic kinetic profiles for substrates comparable to those derived using detergent solubilized enzyme. Changing the phospholipid composition of the nanodisc had no bearing on the ability of COX-2 to oxygenate AA or to be inhibited by nonselective NSAIDs or celecoxib. The use of nanodisc-reconstituted COX-2 opens up a wide range of solution-based experiments that were otherwise intractable given the presence of detergent. [Learn more]

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The application of nanodisc technology to study COX-2 catalysis and inhibition. (A) Size-exclusion chromatography profile of nanodisc-reconstituted COX-2 (solid line) versus empty nanodisc (dashed line). (B) Representative negative-stain electron micrograph of nanodisc-reconstituted COX-2. (C) 2D k-means class averages of nanodisc-reconstituted COX-2 particles. (D) Model of COX-2 bound to one leaflet of the nanodisc scaffold. (E) Products formed from AA incubation with nanodisc-reconstituted COX-2.
 
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