Bruner group research

Natural Product Biosynthesis


Nonribosomal peptide and polyketide natural products are assembled on large, multidomain and multimodule megasynthase enzymes. The amino acid building blocks are polymerized through a thioester-templated mechanism. The rational, linear organization of these systems favors engineering approaches toward the production of novel compounds. The lack of detailed structural and mechanistic information for multidomain synthases hinders the mechanistic understanding and rational engineering of these systems.  In particular, the structural dynamics of the carrier domains and the details of substrate trafficking remain largely undetermined.
 
We are applying a structural genomics approach to natural product biosynthetic gene clusters. The study of individual components of a complex biosynthetic pathway provides an important but limited picture of the overall pathway. A broad focus on the structure and function of all components will provide valuable information on concerted function and assist in engineering efforts. To accomplish these goals, we apply a largely chemical approach, using synthetic inhibitors and analogs to provide detailed insights into the chemistry. 

Mechanistic Enzymology

 

The complex structure and functionality of therapeutically important natural products is often the result of interesting and unusual enzymology. For example, the enzyme DpgC catalyzes a key step in the biosynthesis several natural products including the vancomycin/teicoplanin antibiotics, important drugs that are frequently used as the last line of defense for aggressive, multidrug-resistant infections. DpgC is a cofactor/metal-independent dioxygenase in the pathway to the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine. The mechanistic details for cofactor-independent oxygenases are unusual and the specific chemistry of DpgC has little precedent.
 
Another example is the aminomutase, SgTAM. beta-Amino acids are important components of a wide range of natural and synthetic compounds. For several classes of nonribosomal peptide natural products, b-amino acid building blocks are generated by 4-methylideneimidazole-5-one (MIO) based aminomutases from the corresponding L-amino acid. Biosynthetic pathways containing an MIO-aminomutase were first described for the antitumor/antibiotic enediynes and later for other classes of natural products including taxanes. For these examples and others, our group has provided insights into structure and mechanism using X-ray crystallography, synthetic inhibitors and mechanistic enzymology.

Natural product discovery

 

In addition we are exploring novel natural product pathways through "genome mining" approaches. For example, we are exploiting a system more amenable to crystallographic analysis, a novel nonribosomal peptide pathway from the thermophile T. fusca. In general, proteins from thermophilic hosts exhibit enhanced stability favoring structural and biochemical characterization. We identified a single gene cluster in T. fusca corresponding to a multimodular nonribosomal peptide secondary metabolite biosynthetic pathway and determined the structure using NMR/MS techniques. The results established a novel structural architecture in the siderophore family of natural products. Current and future work is aimed at structurally characterizing all enzymes in this pathway to provide a complete structural basis for nonribosomal peptide natural product biosynthesis.