Prenyltransferases catalyze alkylation of carbon-carbon double bonds, aromatic rings, amino groups, hydroxyl groups, and thiol groups by allylic isoprenoid diphosphate esters. These are major building and modification reactions required for the synthesis of over 55,000 naturally occurring molecules. Prenyltransferases are unique to the isoprenoid biosynthetic pathway, where they catalyze the major building reactions required to construct isoprenoid carbon skeletons from simple five-carbon building blocks and the addition of isoprene units to non-isoprenoid molecules. In nature these compounds function as hormones, as structural components in membranes, as lipophilic moieties that anchor cofactors and proteins to membranes, in vision, as mating pheromones, and as defensive agents, to name a few. They are used to treat cancer and regulation of isoprenoid metabolism is the basis for highly successful treatments of coronary heart disease. The long-term goals of this project are to determine the mechanisms of prenyl transfer reactions, to understand how prenyltransferases catalyze these reactions, to understand how changes in the structures of prenyltransferases lead to the synthesis of new products, and to use this knowledge to study the biological properties of isoprenoid molecules and develop new applications for biotechnology. To facilitate this work we are developing a library of E. coli strains genetically engineered to produce prenyltransferases for use in our laboratory and by collaborators for X-ray studies, procedures to synthesize substrates, substrate analogs and inhibitors, and new assays for enzyme activity. Our strains, protocols and molecules are widely used by other workers in the field. In addition to mechanistic work with protein farnesyltransferase, the enzyme is being harnessed to study the interactions between proteins posttranslationally modified with isoprenoid moieties and cellular membranes by single-molecule detection of fluorescently tagged farnesylated peptides at lipid-coated surfaces. In addition, protein farnesyltransferase is being used to region-selectively modify proteins with analogs of farnesyl diphosphate bearing bio-orthogonal functional groups. The modified proteins can be chemoselectively conjugated with non-protein molecules or surfaces. We are developing applications for covalently attaching proteins, including antibody-binding proteins, to silica and gold surfaces and for conjugating proteins with polyethylene glycol.

Public Health Relevance

Coronary heart disease and cancer are the two leading causes of death in the United States. Chemical regulation of sterol metabolism with the statin family of cholesterol lowering compounds is the basis of the recently developed and highly successful treatment of heart disease. Naturally occurring isoprenoid molecules, or molecules patterned after the natural compounds, are clinically approved and highly successful drugs for the treatment of cancer. Research funded by this proposal studies the enzyme catalysts that construct the basic carbon skeletons of isoprenoid molecules and the development of biotechnological applications based these enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021328-38
Application #
8532677
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
1977-06-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
38
Fiscal Year
2013
Total Cost
$420,355
Indirect Cost
$135,560
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Choi, Seoung-Ryoung; Seo, Jin-Soo; Bohaty, Rochelle F H et al. (2014) Regio- and chemoselective immobilization of proteins on gold surfaces. Bioconjug Chem 25:269-75
Rudolf, Jeffrey D; Poulter, C Dale (2013) Tyrosine O-prenyltransferase SirD catalyzes S-, C-, and N-prenylations on tyrosine and tryptophan derivatives. ACS Chem Biol 8:2707-14
Rudolf, Jeffrey D; Wang, Hong; Poulter, C Dale (2013) Correction to multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase. J Am Chem Soc 135:10879
Seo, Jin-soo; Lee, Sungwon; Poulter, C Dale (2013) Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays. J Am Chem Soc 135:8973-80
Rudolf, Jeffrey D; Wang, Hong; Poulter, C Dale (2013) Multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase. J Am Chem Soc 135:1895-902
Heaps, Nicole A; Poulter, C Dale (2011) Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase. J Org Chem 76:1838-43
Pan, Jian-Jung; Bugni, Tim S; Poulter, C Dale (2009) Recombinant squalene synthase. synthesis of cyclopentyl non-head-to-tail triterpenes. J Org Chem 74:7562-5
Thulasiram, Hirekodathakallu V; Erickson, Hans K; Poulter, C Dale (2008) A common mechanism for branching, cyclopropanation, and cyclobutanation reactions in the isoprenoid biosynthetic pathway. J Am Chem Soc 130:1966-71
McDougal, Owen M; Turner, Matthew W; Ormond, Andrew J et al. (2008) Three-dimensional structure of conotoxin tx3a: An m-1 branch peptide of the M-superfamily. Biochemistry 47:2826-32
Thulasiram, Hirekodathakallu V; Erickson, Hans K; Poulter, C Dale (2007) Chimeras of two isoprenoid synthases catalyze all four coupling reactions in isoprenoid biosynthesis. Science 316:73-6

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