"Fighting Fascism in Europe. The World War II Letters of an American Veteran of the Spanish Civil War," by Lawrence Cane

 

David E. Cane

 

Research

Mechanistic Enzymology of Natural Products Biosynthesis

The main focus of our research has been to establish the mechanism of formation of a wide variety of natural products of diverse biological origin. These metabolites include antibiotics, toxins, plant defense substances, essential oils, and vitamins. Over the last several years our research group has concentrated on the mechanistic enzymology and molecular genetics of two broad areas, terpenoid metabolism and polyketide antibiotic biosynthesis.

Our studies of terpenoid synthesizing enzymes have focused in recent years on determination of the structure of these proteins and in the use of various genetic and chemical tools to elucidate the mode of action of these enzymes.  One of the great challenges in genomics and proteomics is the identification of the biochemical function of the thousands of uncharacterized gene products that are filling the emerging genome sequence databases.  Using an approach that can be called “genome mining” we have been expressing and characterizing the component genes of terpenoid biosynthetic gene clusters in Streptomyces species that play an important role in microbial genetics and serve as the sources of majority of known antibiotic natural products.  In the course of this work we continue to discover previously unknown enzymes and to identify their mode of action.  For example we have now defined the role of all the genes encoding biosynthesis of the newly discovered metabolite, neopentalenolactone, including a new cyctochrome P450, two new non-heme iron-dependent dioxygenases, and a new flavin-dependent Baeyer-Villiger monoxygenase. We have also discovered a completely unanticipated mechanism for the enzymatic formation of geosmin, the ubiquitous microbial metabolite responsible for the characteristic odor of soil and the source of the “off-taste” of water and various foods, as well as an entirely new pathway for the biosynthesis of a second “off-taste” constituent, methyl isoborneol. In collaborative research with Prof. David Christianson of the University of Pennsylvania, we have continued to determine several new terpenoid synthase structures of both native and mutant enzymes, either free or with bound substrates or inhibitors.

Investigations in our lab of the genetics and enzymology of polyketide antibiotic biosynthesis include the study of the antibiotics erythromycin, picromycin, methymycin and tylosin, as well as the polyether antibiotic nanchangmycin (dianemycin). Most of this effort has been carried out in close collaboration with the group of Prof. Chaitan Khosla of Stanford University, and more recently with Prof. Adrian Keatinge-Clay of the University of Texas.  Recent major accomplishments include the development of a sensitive and robust method to determine the stereochemical specifity of the fundamental polyketide chain building reaction, based on the dissection of several modular synthases into their functional component domains and reconstitution of the activity of the complete modules, and the analysis of the structure and biochemical properties of dissected polyketide synthase modules. The heretofore intrinsic epimerase activity of specific ketoreductase domains has been elucidated using a newly developed equilibrium isotope exchange assay. Recently we have also shown using a tandem equilibrium isotope exchange assay that certain redox-inactive ketoreductase-like domains, known as KR(0) domains, possess a cryptic yet essential epimerase activity. In related studies we have been elucidating the mechanism, substrate specificity, and stereochemistry of dehydrations catalyzed by PKDS dehydratase domains.