Isoprenoid compounds comprise the largest family of small molecules in nature. Many are essential for cells to survive and elements of the isoprenoid biosynthetic pathway are found in all organisms. Several isoprenoid biosynthetic enzymes are targets for drugs. Lipitor, the most widely prescribed drug, targets an enzyme early in the pathway to lower serum cholesterol. Fosamax, used to treat osteoporosis, targets a chain elongation enzyme in the middle of the pathway. The antifungal drug Lamisil targets the later stages of fungal steroid biosynthesis. Differences between the isoprenoid biosynthetic pathways and enzymes in humans and microorganisms present new attractive targets for drug development. In addition, products of the pathway have a long history as drugs, including more recent examples such as taxol (cancer) and artemisinin (malaria). This project supports research to elucidate new biosynthetic routes to isoprenoid compounds and structural and mechanistic studies of enzymes in the pathways from where the routes diverge from fatty acid metabolism and glycolysis up to the synthesis of the two fundamental five-carbon building blocks of isoprenoid molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The work incorporates a combination of genetics, molecular biology, mechanistic enzymology, structural biology, and synthetic organic chemistry in this effort. The PI's laboratory is a unique site for this project. He and his coworkers have expertise specifically related to the isoprenoid pathway in the diverse areas of research outlined above. His laboratory has a large collection of bacterial and fungal clones bearing knockouts or additions of appropriate biosynthetic enzymes, expression vectors for production of purified enzymes, substrates/alternate substrates/inhibitors, and the analytical tools required for identification of new metabolites and for structural and mechanistic studies. Three interrelated areas of research are proposed for the coming grant period. (1) All available evidence indicates that most, if not all, Archaea synthesize isopentenyl diphosphate from mevalonate differently that eukaryotes. Specifically, mevalonate phosphate is first converted to isopentenyl phosphate (IP) and then to the diphosphate. The gene for synthesis of IP is missing. Identification of the gene and verification of its function will be undertaken. Related work on X-ray structure-function and mutagenesis studies of IP kinase will be conducted, including the development of mutants capable of efficiently synthesizing longer chain diphosphates from the corresponding phosphates. (2) Isoforms of IPP isomerase (IDI1 and IDI2) convert IPP to DMAPP by a proton-initiated process. NMR, X-ray, UV, and fluorescence experiments will be conducted to identify the amino acids/cofactors involved in the proton transfer reactions, including the unusual role of reduced flavin in the proton transfer reactions of IDI2. (3) Amine and thiol-containing analogues of methylerythritol phosphate and hydroxydimethylallyl diphosphate will be synthesized and examined as mechanism-based inhibitors of the iron-sulfur cluster enzymes the methylerythritol phosphate pathway.

Public Health Relevance

The isoprenoid biosynthetic pathway synthesizes over 60,000 natural products, many of which such as taxol and artemisinin, are valuable drugs for treatment of human disease. Furthermore, metabolites from the biosynthetic are essential for the survival of all organisms. This proposal focuses on the genes and enzymes for the early steps in the isoprenoid pathway. Results from this study are important for developing procedures for in vivo synthesis of complex natural and unnatural molecules that are difficult to synthesize by chemical means. Inhibition of early enzymes in the pathway are important targets. We are developing technology for high throughput screening technology to target antibiotic and antiparasitic drug candidates that target the isoprenoid pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025521-33
Application #
8323900
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
1979-04-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
33
Fiscal Year
2012
Total Cost
$389,543
Indirect Cost
$126,583
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
Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E et al. (2014) Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens. J Org Chem 79:9170-8
de Ruyck, Jerome; Schubert, Heidi L; Janczak, Matthew W et al. (2014) Tetartohedral twinning in IDI-2 from Thermus thermophilus: crystallization under anaerobic conditions. Acta Crystallogr F Struct Biol Commun 70:347-9
de Ruyck, Jerome; Janczak, Matthew W; Neti, Syam Sundar et al. (2014) Determination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae. Chembiochem 15:1452-8
Janthawornpong, Karnjapan; Krasutsky, Sergiy; Chaignon, Philippe et al. (2013) Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the methylerythritol phosphate pathway. J Am Chem Soc 135:1816-22
Mabanglo, Mark F; Serohijos, Adrian W R; Poulter, C Dale (2012) The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for archaeal isopentenyl phosphate kinase. Biochemistry 51:917-25
Ahrens-Botzong, Annegret; Janthawornpong, Karnjapan; Wolny, Juliusz A et al. (2011) Biosynthesis of isoprene units: Mossbauer spectroscopy of substrate and inhibitor binding to the [4Fe-4S] cluster of the LytB/IspH enzyme. Angew Chem Int Ed Engl 50:11976-9
Heaps, Nicole A; Poulter, C Dale (2011) Type-2 isopentenyl diphosphate isomerase: evidence for a stepwise mechanism. J Am Chem Soc 133:19017-9
Sharma, Nagendra K; Pan, Jian-Jung; Poulter, C Dale (2010) Type II isopentenyl diphosphate isomerase: probing the mechanism with alkyne/allene diphosphate substrate analogues. Biochemistry 49:6228-33
Mabanglo, Mark F; Schubert, Heidi L; Chen, Mo et al. (2010) X-ray structures of isopentenyl phosphate kinase. ACS Chem Biol 5:517-27
Poulter, C Dale (2009) Bioorganic chemistry. A natural reunion of the physical and life sciences. J Org Chem 74:2631-45

Showing the most recent 10 out of 59 publications