Isonitrilase is involved in constructing a triple bond from a primary amine and a carbonyl moiety of carbohydrate phosphate. Non-heme iron and 2-(oxo)glutarate-dependent (Fe/2OG) enzymes catalyze a bewildering array of transformations, which include halogenations, cyclizations, dehydrogenations, endoperoxidation and stereoinversion of aliphatic carbon centers. In more than 40 isolated vinyl isonitrile containing natural products, a universal approach involving a consecutive two-step enzymatic transformation by an isonitrilase and an Fe/2OG dependent alkene forming enzyme is proposed to install these functional groups. However, the mechanistic understating of these transformations remains to be elucidated. A chemical and biosynthetic understanding of these reaction mechanisms could enable reprograming of isonitrilase and Fe/2OG enzymes as biocatalysts for producing new compounds with improved bioactivity. Having recently made progress toward Fe/2OG enzyme catalyzed epoxidation and desaturation, and isonitrilase activity reconstitution in vitro, we propose to study the isonitrile and decarboxylation-assisted alkene formation reactions on the pathway to rhabduscin and paerucumarin biosyntheses. We will use an integrated approach to provide molecular understanding of the enzyme mechanisms and, in select cases, evaluate the substrate flexibility and reprogram the related enzymes by directed evolution.

Public Health Relevance

Isonitrile and alkene functionalities are widely distributed in many important natural products with antibacterial and anticancer properties. The biosynthetic and chemical logic for these functional groups installation are not in the repertoire of current synthetic chemistry. The molecular understating of these enzymatic transformations elucidated in this project would enable designing of these enzymes as biocatalysts and programming biosynthetic pathways to make new compounds with improved bioactivities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM127588-02
Application #
9700698
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2018-06-01
Project End
2023-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
North Carolina State University Raleigh
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042092122
City
Raleigh
State
NC
Country
United States
Zip Code
27695