The goal of this project is to determine the biosynthetic steps leading to the production of ladderanes, a class of unusual bacterial lipids displaying a highly strained structure composed of three to five linearly fused cyclobutane rings. The biosynthesis of ladderanes is thought to involve enzymes belonging to the radical SAM enzyme superfamily including several cobalamin-dependent radical SAM enzymes. Cobalamin- dependent radical SAM enzymes are also involved in the biosynthesis of a number of pharmaceutically important compounds including anti-microbial, anti-tumor, and anti-viral (including anti-HIV) agents. However, the mechanisms by which these enzymes carry out their unusual chemical transformations remain unknown to date. The knowledge gained from an understanding of the steps involved in ladderane biosynthesis may, therefore, lead to the rational design of pharmaceutical agents with new or improved biological function through the manipulation of these unprecedented reactions. Furthermore, ladderane producing bacteria have recently ben utilized for the bioremediation of nitrogen contaminated wastewater. A detailed understanding of ladderane biosynthesis may provide new insight for the development and bioengineering of this industrial process, which would be a boon for public health. To accomplish this goal, the enzymes involved in the biosynthesis of ladderanes will be recombinantly expressed, purified, and assayed for activity in the production of lipid biosynthetic intermediates. The identity and structural determination of the biosynthetic pathway intermediates will be achieved using high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy by comparison with synthetic standards. The precise mechanisms underlying the activity of the cobalamin-dependent radical SAM enzymes involved in ladderane biosynthesis will be interrogated using deuterated substrates to determine the stereochemistry of the proposed hydrogen atom transfers and a combination of UV/Vis and electron paramagnetic resonance spectroscopy to determine the relevant redox states of the cobalamin cofactor during catalysis.

Public Health Relevance

The goal of this research is to study the enzymes involved in the production of ladderanes, an unusual class of bacterial lipids. Several of the enzymes involved in the production of ladderanes are similar to those involved in the production of a number of pharmaceutically important compounds including anti-microbial, anti-tumor, and anti-viral (including anti-HIV) agents. The way in which these enzymes carry out their functions remains unknown and a detailed understanding of the enzymes responsible for ladderane production has the potential to lead to the development of new pharmaceuticals.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM103181-02
Application #
8531002
Study Section
Special Emphasis Panel (ZRG1-F04-D (20))
Program Officer
Lees, Robert G
Project Start
2012-07-25
Project End
2014-07-24
Budget Start
2013-07-25
Budget End
2014-07-24
Support Year
2
Fiscal Year
2013
Total Cost
$52,190
Indirect Cost
Name
University of Texas Austin
Department
None
Type
Schools of Pharmacy
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712