Microbial natural products possess complex chemical structures as well as potent biological activity and are an important source of drugs. While these molecules have captivated synthetic and medicinal chemists for decades, more recently the underlying biosynthetic pathways that construct natural product scaffolds have been recognized as important reservoirs of novel enzymes. Uncovering new enzymatic chemistry and biosynthetic strategies expands our basic understanding of Nature?s synthetic capabilities. It is also a critical first step toward applications of this fundamental knowledge and can serve as an inspiration for synthetic chemists. The long- term goal of the proposed research is to identify microbial enzymes that catalyze previously unappreciated chemical transformations. We envision discovering such enzymes by studying the biosynthesis of natural products containing important molecular architecture and functional groups of unknown biosynthetic origin. An important class of such structural motifs are functional groups containing a nitrogen-nitrogen (N?N) bond, a chemical linkage found in 9% of the 200 best-selling drugs. Reactive N?N bond-containing functional groups, including diazo and N-nitroso groups, are a critical part of biologically active small molecules including streptozotocin (Zanosar), a clinically used treatment for metastatic pancreatic cancer. They are also uniquely enabling chemical reagents, with diazo compounds mediating many important and powerful chemical transformations in synthetic chemistry, biocatalysis, and biorthogonal chemistry. Though reactive N?N bonds are present in microbial natural products, their biosynthetic origins are poorly understood. Thus, the overall objective of this application is to discover and characterize enzymes that construct diazo- and N-nitroso- containing metabolites. Preliminary results from our lab and others have uncovered biosynthetic gene clusters responsible for constructing multiple diazo- and N-nitroso-containing natural products, including streptozotocin and other molecules that have been in clinical trials. These findings set the stage for our three complementary specific aims: 1) identify and characterize the biosynthetic enzymes responsible for constructing the diazo groups of the natural products cremeomycin and kinamycin; 2) identify and characterize the biosynthetic enzymes responsible for constructing the N-nitroso groups of the natural products streptozotocin and alanosine; 3) access additional diazo and N-nitroso biosynthetic enzymes and natural products by characterizing cryptic gene clusters. By leveraging the tremendous structural diversity of microbial natural products in the genomic era, we will rapidly discover and characterize biosynthetic transformations that fill critical gaps in our current knowledge of enzymatic chemistry capabilities. Finally, the workflow we have formulated for investigating the biosynthesis of reactive N? N bond-containing functional groups will also be readily generalizable to additional structural motifs found in microbial natural products.

Public Health Relevance

Small molecules produced by microorganisms are an important source of life-saving drugs, and the protein- based catalysts (enzymes) that build these molecules can perform unusual chemical reactions. The goal of this proposal is to discover enzymes that construct reactive functional groups containing nitrogen?nitrogen (N?N) bonds, structural features that are found in many biologically active molecules, including approved drugs used to treat cancer. By discovering and characterizing enzymes that build N?N bond-containing diazo and N-nitroso groups, we will expand our fundamental understanding of biological chemistry and inform future applications toward improving human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Bond, Michelle Rueffer
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Harvard University
Schools of Arts and Sciences
United States
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