Mechanistic Studies On Three Organophosphonate-processing Enzymes, Hppe, Hepd And Mpns

Open Access
Wang, Chen
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
October 24, 2014
Committee Members:
  • Carsten Krebs, Dissertation Advisor
  • Joseph M Bollinger Jr., Committee Chair
  • Squire J Booker, Committee Member
  • Michael Thomas Green, Committee Member
  • James Homer Tumlinson Iii, Committee Member
  • Organophosphonate
  • mechanism
  • HppE
  • HEPD
  • MPnS
  • non-heme-iron dependent enzyme
Naturally occurring phosphonates and phosphinates have bioactivities (e.g., herbicidal, antibiotic) that are useful in agriculture and medicine. Phosphonate and phosphinate compounds can potently inhibit enzymes in various metabolic pathways by functioning as stable mimics of phosphate esters and carboxylic acids. Biosynthetic pathways to phosphonate and phosphinate compounds have proven to be treasure troves for the discovery of unusual enzymatic reactions. The investigation of these conserved pathways has revealed three unprecedented biochemical steps catalyzed by the non-heme-iron(II) enzymes, HppE [(S)-2-hydroxypropyl-1-phosphonate epoxidase], HEPD (2-hydroxyethylphosphonate dioxygenase) and MPnS (methylphosphonate synthase). The work described herein focused on understanding both the mechanisms of the individual reactions and the structural/functional features of each enzyme important in specifying its reaction and pathway. The iron-dependent epoxidase, HppE, converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic, fosfomycin [(1R, 2S)-epoxypropylphosphonate], in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE had been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (H•) from C1 of S-HPP to initiate epoxide ring closure. In my work, we showed that the preferred co-substrate is actually H2O2 and that HppE therefore almost certainly employs an iron(IV)-oxo complex as the H• abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 showed that reduction of O2 and utilization of the resultant H2O2 were actually operant. The mechanism of the conversion of 2-hydroxyethylphosphonate to hydroxymethylphosphonate (2-HEP) catalyzed by iron-dependent enzyme, HEPD during the biosynthesis of the commercial herbicide, phosphinothricin, had been enigmatic. By using rapid-kinetic and spectroscopic methods, we detected an iron(IV)-oxo (ferryl) intermediate in the HEPD reaction. Kinetic analysis suggested that the intermediate is kinetically competent to be on the productive pathway. The accumulation of this intermediate only with substrate having deuterium in the abstracted pro-S position of C2 of 2-HEP implied that the ferryl intermediate abstracts this hydrogen, but the increased accumulation of the ferryl complex in 2H2O solvent implied that the hydrogen becomes solvent-exchangeable before the ferryl abstracts it. To account for these unanticipated results, a mechanism involving initial abstraction of the pro-S hydrogen by an Fe(III)-superoxo precursor to the ferryl complex, transfer of a hydroxyl group containing the originally abstracted hydrogen to C2 concomitant with formation of the ferryl complex, and an unprecedented abstraction of H• from the newly installed C2 OH group by the ferryl complex was proposed. Like HEPD, the iron-dependent oxygenase, MPnS, also catalyzes the 4e--oxidative C-C cleavage of 2-HEP, but generates different products, methylphosphonate and CO2. MPnS, HEPD, and HppE have quite striking structural similarity and utilize identical or similar phosphonate substrates, but they employ different oxidants, O2 or H2O2, to effect three completely different reactions. By using H2O2, HppE effects a 2e- oxidation (epoxide installing 1,3-dehydrogenation) of S-HPP. HEPD and MPnS catalyze distinct 4e--oxidative C1-C2-cleaving transformations of 2-HEP, in each case with O2 as the oxidant. We explored the distinct but potentially overlapping catalytic capabilities of the three enzymes on the two different phosphonate substrates with the two different oxidants. As expected from its reassignment as a peroxidase, HppE fails to catalyze a 4e--oxidative C-C cleavage reaction with O2 as oxidant. However, we found that both MPnS and HEPD can catalyze the 2e- dehydrogenation of 2-HEP with H2O2 rather than O2 as the oxidant. HEPD catalyzed only a fraction of a turnover under the conditions examined, consistent with the fact that it is an oxygenase. By contrast, MPnS, also a known oxygenase, surprisingly catalyzed up to 25 turnovers of 2-HEP to the corresponding aldehyde with H2O2 as the oxidizing co-substrate. The physiological relevance of this activity is unknown. In sum, all three enzymes possess some peroxidase activity, with HppE being by far the most efficient, but only HEPD and MPnS exhibit the 4e--oxidative C-C-cleavage activity.