Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads
Abstract
The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy) and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 performs the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate but instead performs an oxidation to make 2-OPP. Additionally, we show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light on the reaction mechanisms of the two terminal enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); Hager Fellowship
- OSTI Identifier:
- 1438868
- Grant/Contract Number:
- PO1 GM077596; 5T32-GM070421
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Chemical Biology
- Additional Journal Information:
- Journal Volume: 12; Journal Issue: 2; Journal ID: ISSN 1554-8929
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Peptides and proteins; Bacteria; Monomers; Chemical structure; Oxygen
Citation Formats
Olivares, Philip, Ulrich, Emily C., Chekan, Jonathan R., van der Donk, Wilfred A., and Nair, Satish K. Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads. United States: N. p., 2016.
Web. doi:10.1021/acschembio.6b00939.
Olivares, Philip, Ulrich, Emily C., Chekan, Jonathan R., van der Donk, Wilfred A., & Nair, Satish K. Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads. United States. https://doi.org/10.1021/acschembio.6b00939
Olivares, Philip, Ulrich, Emily C., Chekan, Jonathan R., van der Donk, Wilfred A., and Nair, Satish K. Tue .
"Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads". United States. https://doi.org/10.1021/acschembio.6b00939. https://www.osti.gov/servlets/purl/1438868.
@article{osti_1438868,
title = {Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads},
author = {Olivares, Philip and Ulrich, Emily C. and Chekan, Jonathan R. and van der Donk, Wilfred A. and Nair, Satish K.},
abstractNote = {The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy) and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 performs the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate but instead performs an oxidation to make 2-OPP. Additionally, we show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light on the reaction mechanisms of the two terminal enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.},
doi = {10.1021/acschembio.6b00939},
journal = {ACS Chemical Biology},
number = 2,
volume = 12,
place = {United States},
year = {Tue Dec 27 00:00:00 EST 2016},
month = {Tue Dec 27 00:00:00 EST 2016}
}
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