The biosynthesis, mechanistic enzymology and molecular genetics of terpenoid biosynthesis in bacteria and fungi will be investigated, with particular emphasis on the mechanistic and structural characterization of three broad classes of proteins uncovered by mining of genomic databases and functional gene expression that present novel structural, mechanistic, or biological features: 1) Investigations of terpene synthases will focus on defining the cyclization mechanisms and elucidating the role of each protein in catalyzing the formation of its individual cyclization products. A combination of bioinformatic tools as well as mutational approaches will be used to identify targets that represent new structural types or unusual biosynthetic reactions and mechanisms, or that correspond to cryptic synthases of currently unknown function that are implicated in biologically important processes such as fungal virulence. The mechanistic focus will be on discovering new cyclases and on unraveling the protein structural basis for catalysis and product specificity using a combination of site-directed mutagenesis and protein crystallography. 2) A variety of catalytically novel oxygenases will be investigated that play key roles in the metabolic conversion of terpene hydrocarbons and alcohols to biosynthetic end-products. 3) The regulation of microbial terpene biosynthesis will also be probed, focusing on two intriguing model systems, the regulation of the biosynthesis of the antibiotic pentalenolactone and the exploration of the cryptic role of a highly conserved nucleotide-binding protein associated with the biosynthetic genes for the widely occurring volatile organic metabolite 2-methylisoborneol. Tens of thousands of terpenoid natural products are present in both terrestrial and marine plants, in fungi and liverworts, and numerous bacteria. Many of these compounds are widely used in human medicine such as the antitumor agent taxol or the antimalarial artemisinin, or harmful to human and animal health, such as the widely occurring trichothecene mycotoxins. Many terpenoids are known plant or fungal hormones, plant protectants, or microbial virulence factors. The ubiquitous volatile bacterial organics geosmin and methylisoborneol, which are responsible for the characteristic odor of moist soil, are also the cause of periodic, widely occurring, and costly episodes of unpleasant taste and odor in public drinking water supplies and in aquaculture. Beyond the intrinsic biological importance of terpenoid metabolites, the study of microbial terpene biochemistry, structural biology, and molecular genetics provides a unique platform for the discovery of new biochemical reactions and mechanisms, for the understanding of the molecular logic of natural product biosynthetic pathways, for the elucidation of the still obscure factors regulating expression of these pathways, and for the coordinated application of biochemical, structural biological, bioinformatic, and molecular genetics tools to the understanding of microbial metabolism.

Public Health Relevance

Terpenoid metabolites, the largest known group of natural products, include many medicinally important substances (taxol for treatment of cancer, artemisin for malaria) as well as substances harmful to human and animal health (trichothecane fungal toxins), in addition to numerous antibiotics, fungal virulence factors, immunosuppresants, neurotoxins, flavor and odor constituents, and natural plant defense compounds. Studies of the biosynthesis of these compounds by bacteria and fungi can not only lead to an understanding of fundamental metabolic pathways, but facilitate the development of practical tools for the generation of new medicinal agents or for prevention of the formation of toxic or otherwise undesirable metabolites by pathogenic or environmental microorganisms. Moreover, mining of genomic information to biochemically characterize cryptic genes of unknown function will lead to fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030301-32
Application #
8451254
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
1982-01-01
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
32
Fiscal Year
2013
Total Cost
$443,789
Indirect Cost
$157,614
Name
Brown University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
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