Distefano Research Group (pdf)

The Distefano Research Group uses synthetic organic chemistry in conjunction with a wide variety of biochemical, spectroscopic and computational techniques to study interesting problems at the interface of chemistry and biology.

Current Projects

The Distefano Research Group has worked in a number of different areas including protein engineering, protein prenylation and cellular patterning.   Currently, our research revolves around isoprenoid diphosphate utilizing enzymes and the projects described below are underway.   Many of these projects involve collaborations with other investigators at the University of Minnesota or at other institutions.

Protein Prenylation:   Protein prenylation consists of the addition of C15 and C20 isoprenoid groups to proteins via the formation of thioether bonds near the C-termini of various   proteins.   Since a large number of proteins are prenylated, and inhibition of prenylation can inhibit the growth of cancer cells, there is considerable interest in developing inhibitors of this protein modification.

Mechanism of the reaction catalyzed by prenyltransferases . Stereochemical and kinetic isotope effect measurements are being used in conjuction with computational modeling to determine the transition state structure of the reaction catalyzed by protein farnesyltransferase.

Figure. TS structure (electrostatic potential) for the reaction catalyzed by PFTase.   The nucleophilic S atom is red with C-1 to the right.

Time resolved X-ray crystallography .   Caged forms of farnesyl diphosphate (FPP) have been synthesized and are being used to observe the reaction as it is catalyzed by protein farnesyltransferase via time resolved crystallography.

Figure. Left: View of structure of ternary complex of PFTase showing long 8.48 Å distance between nucleophile and electrophile.   Right: Caged isoprenoids and peptide.

            Interactions between prenylated proteins .   The interaction between cdc42 (a prenylated protein) and Rho GDI is being studied.   Prenylated peptides that bind to Rho GDI have been synthesized and their interaction with Rho GDI has been studied in vitro and in vivo.

Figure .   A confocal microscope image of Hela cells incubated for 1h in the presence of 1 μM C7-2c (a geranylgeranylated peptide containing a fluorescein label and a penetratin domain for cell penetratin) . This peptide localizes in a punctuate fashion around the nucleus.   This image shows the fluorescence due to the prenylated peptide superimposed on a bright field microscope image.

            Selective protein labeling .   We have developed a method that can be used to incorporate azide and alkyne labels into proteins at a unique position near the C-terminus.   We are exploiting this to examine the specificity of prenyltransferases as well as to immobilize and selectively label proteins.  

Figure . (E) Immobilization strategy using eGFP incorporating a PFTase "CAAX" box recognition sequence showing initial prenylation with C6-3 followed by capture using azide-functionalized agarose beads.   (F) Model of eGFP-Texas Red conjugate obtained by enzymatically incorporating alkyne C6-3 followed by reaction with Texas-Red-azide.

Rubber Biosynthesis:   Rubber, isolated from natural latex, is a 20 million ton world industry.   It is used in the manufacture of automobile and aircraft tires as well as in the production of more specialized products such as surgical gloves and angioplasty balloons.   At present, all natural latex is isolated from a single plant species, Hevea brasiliensis that does not grow in North America. The goal of our research is to identify the proteins and clone the genes responsible for rubber production in the rubber tree, Hevea brasiliensis .   If this can be accomplished, it will set the stage for creating new rubber producing plants that grow in the United States or allow the upregulation of rubber production in indigenous plants the already produce rubber but at levels that are not commercially viable.

            Photoaffinity labeling of rubber producing enzymes .   A number of analogues of farnesyl diphosphate (FPP) that incorporate photoactivatable groups have been synthesized.   Upon photolysis, in the presence of biosynthetically active rubber particles, these compounds crosslink with proteins involved in rubber synthesis.   Using this approach, several proteins have been identified.

Figure: Right: Chemical probes that will be used in the functional identification of rubber biosynthetic enzymes.   Left: SDS PAGE analysis of photoaffinity labeling reactions of WRPs isolated from Hevea brasiliensis using compound 1-1 .   Lanes 1 and 2, gel visualized with Sypro-Orange; Lanes 1' and 2', same gel visualized by phosphorimaging to detect radioactivity. Lane 1: Reaction containing WRPs and compound 1-1 (10 µM).   Lane 2: Reaction containing WRPs, compound 1-1 and FPP (100 µM).   Photolysis was performed at 4°C for 6 h followed by separation via SDS PAGE.

Illudin Biosynthesis: Higher basidiomycetes are known to produce a variety of sesquiterpenes with unique skeletons not found in microbes or plants as a strategy to deter feeding on their fruiting bodies. One class of these sesquiterpenoids, the illudanes, has received considerable attention because of their potent antitumor and antibacterial activity. Initially discovered as antibiotic substances from the Jack O'Lantern mushroom Omphalotus olearius , these compounds were later identified as the sesquiterpenes illudin M and S. Illudin S exhibits cytotoxic and cytostatic properties at nanomolar concentrations in several human tumor cell lines, including multiple drug resistant tumors.

            Identification and mechanistic analysis of the cyclase responsible for the formation of ∆6-protoilludene .   Several photoaffinity labeling analogues (similar to those used in the rubber project described above) and mechanism based inactivators are being prepared to study the cyclization reaction (shown below) that converts farnesyl diphosphate to ∆6-protoilludene.

Figure . Cyclization of FPP to ∆6-protoilludene and probes used to identify the enzyme and probe the specificity and mechanism.