The ultimate goal of this project is to understand at the molecular level how the oxirane group of fosfomycin, the cyclohexene moiety of spinosyn A, and the annelated oligocyclobutane ring system of ladderane lipids are biosynthesized. Fosfomycin is a clinically useful antibiotic and chemically versatile synthon in the research and development of new therapeutics largely on account of its reactive epoxide ring. Spinosyn A is an environmentally """"""""green"""""""" commercial insecticide with activity against insects detrimental to both human health and harvested plants. Ladderane lipids are a necessary component of the intracytoplasmic compartment of bacteria responsible for anaerobic ammonia oxidation (anammox). This process is employed in the bioremediation of nitrogen-contaminated wastewater. Therefore, the continued development of these compounds in the interest of public health requires a more complete understanding of the chemistry underlying their biosyntheses. To achieve these goals, detailed enzyme analysis will be coupled with chemical synthetic methods to develop mechanistic probes specific to the unique challenges each system presents. The mononuclear non-heme iron enzyme HppE, which is responsible for the oxirane of fosfomycin, will be investigated using a combination of 18O kinetic isotope effects, radical clock probes, designer substrate analogues, and spectroscopic methods. These techniques will be used to characterize the radical intermediates of HppE catalysis and to identify the reactive iron-oxygen species (FeIII-OO? versus FeIV=O) responsible for their formation. The enzyme SpnF, which catalyzes the [4+2]-cycloaddition responsible for the construction of the cyclohexene ring of spinosyn A, will be investigated to verify whether it is indeed the first- confirmed natural Diels-Alderase. This hypothesis will be tested using secondary deuterium kinetic isotope effects and further characterized via thermodynamic and kinetic study of the cycloaddition reaction. The biosynthetic pathway of ladderanes is believed to involve polyunsaturated fatty acids with cyclization proceeding through B12-dependent radical SAM chemistry. Therefore, the putative biosynthetic gene cluster from Kuenenia stutgartiensis will be interrogated by reconstituting the ful pathway in vitro using chemo- enzymatically prepared substrates so as to characterize the key cyclization reactions. These studies will significantly enhance our understanding of the diverse chemistry and enzymology (non-heme iron-dependent oxidases, B12-dependent radical SAM enzymes, Diels-Alderases) of biological cyclization reactions, which can be exploited in future combinatorial biosynthetic endeavors to generate novel and structurally diverse compounds with therapeutic potential.

Public Health Relevance

The ring containing compounds at the center of the proposal have established relevance with regard to public health in terms of antimicrobial development (fosfomycin), the control of insect pests (spinosyn A) and the bioremediation of nitrogen-contaminated wastewater (the ladderanes). Nevertheless, the biochemistry by which they are produced cannot be understood in terms of currently recognized paradigms of enzyme catalysis. This gap in understanding will be addressed by the proposal such that these and related compounds may be further developed for biomedical and biotechnological purposes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BCMB-U (02))
Program Officer
Gerratana, Barbara
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Texas Austin
Schools of Pharmacy
United States
Zip Code
Yang, Zhongyue; Yang, Song; Yu, Peiyuan et al. (2018) Influence of water and enzyme SpnF on the dynamics and energetics of the ambimodal [6+4]/[4+2] cycloaddition. Proc Natl Acad Sci U S A 115:E848-E855
Besandre, Ronald; Liu, Hung-Wen (2018) Biochemical Basis of Vosevi, a New Treatment for Hepatitis CPublished as part of the Biochemistry series ""Biochemistry to Bedside"". Biochemistry 57:479-480
Ruszczycky, Mark W; Zhong, Aoshu; Liu, Hung-Wen (2018) Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes. Nat Prod Rep 35:615-621
Ko, Yeonjin; Wang, Shao-An; Ogasawara, Yasushi et al. (2017) Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A. Org Lett 19:1426-1429
Jeon, Byung-Sun; Ruszczycky, Mark W; Russell, William K et al. (2017) Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A. Proc Natl Acad Sci U S A 114:10408-10413
Thibodeaux, Christopher J; Liu, Hung-Wen (2017) The type II isopentenyl Diphosphate:Dimethylallyl diphosphate isomerase (IDI-2): A model for acid/base chemistry in flavoenzyme catalysis. Arch Biochem Biophys 632:47-58
Zhang, Qingbo; Li, Huixian; Yu, Lu et al. (2017) Characterization of the flavoenzyme XiaK as an N-hydroxylase and implications in indolosesquiterpene diversification. Chem Sci 8:5067-5077
Lin, Chia-I; McCarty, Reid M; Liu, Hung-Wen (2017) The Enzymology of Organic Transformations: A Survey of Name Reactions in Biological Systems. Angew Chem Int Ed Engl 56:3446-3489
Jeon, Byung-Sun; Wang, Shao-An; Ruszczycky, Mark W et al. (2017) Natural [4 + 2]-Cyclases. Chem Rev 117:5367-5388
Patel, Ashay; Chen, Zhuo; Yang, Zhongyue et al. (2016) Dynamically Complex [6+4] and [4+2] Cycloadditions in the Biosynthesis of Spinosyn A. J Am Chem Soc 138:3631-4

Showing the most recent 10 out of 65 publications