Mechanistic consequences of chiral radical clock probes: Analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates

Hui Huang, Wei Chen Chang, Geng Min Lin, Anthony Romo, Pei Jing Pai, William Russell, David H. Russell, Hung Wen Liu

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.

Original languageEnglish (US)
Pages (from-to)2944-2947
Number of pages4
JournalJournal of the American Chemical Society
Volume136
Issue number8
DOIs
StatePublished - Feb 26 2014
Externally publishedYes

Fingerprint

Clocks
Iron
Enzymes
Substrates
Acids
Enantiomers
Chirality
Biosynthesis
Antibiotics
Fosfomycin
Keto Acids
Stereoisomerism
Electrons
Epoxy Compounds
Processing
2-hydroxypropylphosphonic acid epoxidase
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Mechanistic consequences of chiral radical clock probes : Analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates. / Huang, Hui; Chang, Wei Chen; Lin, Geng Min; Romo, Anthony; Pai, Pei Jing; Russell, William; Russell, David H.; Liu, Hung Wen.

In: Journal of the American Chemical Society, Vol. 136, No. 8, 26.02.2014, p. 2944-2947.

Research output: Contribution to journalArticle

Huang, Hui ; Chang, Wei Chen ; Lin, Geng Min ; Romo, Anthony ; Pai, Pei Jing ; Russell, William ; Russell, David H. ; Liu, Hung Wen. / Mechanistic consequences of chiral radical clock probes : Analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates. In: Journal of the American Chemical Society. 2014 ; Vol. 136, No. 8. pp. 2944-2947.
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abstract = "(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.",
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T2 - Analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates

AU - Huang, Hui

AU - Chang, Wei Chen

AU - Lin, Geng Min

AU - Romo, Anthony

AU - Pai, Pei Jing

AU - Russell, William

AU - Russell, David H.

AU - Liu, Hung Wen

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N2 - (S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.

AB - (S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.

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