Abstract
When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6479-6488 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 57 |
| Issue number | 46 |
| DOIs | |
| State | Published - Nov 20 2018 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
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α-Amine Desaturation of d -Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l -Arginine 3-Hydroxylase, VioC. / Dunham, Noah P.; Mitchell, Andrew J.; Del Río Pantoja, José M.; Krebs, Carsten; Bollinger, Jr., Joseph M.; Boal, Amie Kathleen.
In: Biochemistry, Vol. 57, No. 46, 20.11.2018, p. 6479-6488.Research output: Contribution to journal › Article
TY - JOUR
T1 - α-Amine Desaturation of d -Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l -Arginine 3-Hydroxylase, VioC
AU - Dunham, Noah P.
AU - Mitchell, Andrew J.
AU - Del Río Pantoja, José M.
AU - Krebs, Carsten
AU - Bollinger, Jr., Joseph M.
AU - Boal, Amie Kathleen
PY - 2018/11/20
Y1 - 2018/11/20
N2 - When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.
AB - When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.
UR - http://www.scopus.com/inward/record.url?scp=85056493724&partnerID=8YFLogxK
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U2 - 10.1021/acs.biochem.8b00901
DO - 10.1021/acs.biochem.8b00901
M3 - Article
C2 - 30403469
AN - SCOPUS:85056493724
VL - 57
SP - 6479
EP - 6488
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 46
ER -