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.

Recent publications on terpenoid synthases:

Jiaoyang Jiang, Charles N. Tetzlaff, Satoshi Takamatsu, Masato Iwatsuki, Mamoru Komatsu, Haruo Ikeda, and David E. Cane, “Genome Mining in Streptomyces avermitilis. A Biochemical Baeyer-Villiger Reaction and Discovery of a New Branch of the Pentalenolactone Family Tree,” Biochemistry 48, 6431-6440 (2009).

Chieh-Mei Wang, Russell Hopson, Xin Lin and David E. Cane  “Biosynthesis of theSesquiterpene Botrydial in Botrytis cinerea.  Mechanism and Stereochemistry of the Enzymatic Formation of Presilphiperfolan-8b-ol,” J. Am. Chem. Soc. 131, 8360-8361 (2009).

Bin Zhao, Li Lei, Dmitry G. Vassylyev, Xin Lin, David E. Cane, Steven L. Kelly, Hang Yuan, David C. Lamb Michael R. Waterman, “Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site,” J. Biol. Chem. 284, 36711-36719 (2009).

Cristina Pinedo, Chieh-Mei Wang, Jean-Marc Pradier, Bérengère Dalmais, Mathias Choquer, Pascal Le Pêcheur, Guillaume Morgant, Isidro G. Collado, David E. Cane, Muriel Viaud, “The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea,” ACS Chem. Biol. 3, 791-801 (2008).

Jiaoyang Jiang, Xiaofei He, and David E. Cane, “Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme,” Nat. Chem. Biol. 11, 711-715 (2007).

Ekaterina Y. Shishova, Luigi Di Constanzo, David E. Cane, and David W. Christianson, “Crystal Structure of Aristolochene Synthase from Aspergillus terreus and the Evolution of Templates for the Cyclization of Farnesyl Diphosphate,” Biochemistry 46, 1941-1951 (2007).

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 and Prof. Zixin Deng of Shanghai Jiaotong University.  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. Application of this methodology has allowed for the first time the determination of the biochemical basis of stereochemical control in polyketide biosynthesis and opened up the study of dehydratase domains.

Recent publications on polyketide synthases:

Chiara R. Valenzano, Rachel J. Lawson, Alice Y. Chen, Chaitan Khosla, and David E. Cane, “The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis,” J. Am. Chem. Soc. 131, 18501-18511 (2009).

Roselyne Castonguay, Chiara R. Valenzano, Alice Y. Chen, Adrian Keatinge-Clay, Chaitan Khosla, and David E. Cane, “Stereospecificity of Ketoreductase Domains 1 and 2 of the Tylactone Modular Polyketide Synthase,” J. Am. Chem. Soc. 130, 11598-11599 (2008).

Roselyne Castonguay, Weiguo He, Alice Y. Chen, Chaitan Khosla, and David E. Cane, “Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase,” J. Am. Chem. Soc.129, 13758-13769 (2007).

Chaitan Khosla, Yinyan Tang, Alice Y. Chen, Nathan A. Schnarr, and David E. Cane, “Structure and Mechanism of the 6-Deoxyerythronolide B Synthase,” Ann. Rev. Biochem. 76, 195-221 (2007).

Yinyan Tang, Chu-Young Kim, Alice Y. Chen, David E. Cane, and Chaitan Khosla, “Structural and Mechanistic Analysis of Protein Interactions in Module 3 of the 6-Deoxyerythronolide B Synthase,” Chem. Biol. 14, 931-943 (2007).

Yinyan Tang, Chu-Young, Kim, Irimpan I. Matthews, Alice Y. Chen, David E. Cane, and Chaitan Khosla, “The 2.7 Å Crystal Structure of a 194-kDa Homodimeric Fragment of the 6-Deoxyerythronolide B Synthase,” Proc. Natl. Acad. Sci. USA, 103, 11124-11129 (2006).